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TITLE: Extracorporeal cellular therapy (ELAD) in severe alcoholic hepatitis: A multinational, prospective, controlled, randomized trialThompson et al.Thompson et al. Severe alcoholic hepatitis (sAH) is associated with a poor prognosis. There is no proven effective treatment for sAH, which is why early transplantation has been increasingly discussed. Hepatoblastoma‐derived C3A cells express anti‐inflammatory proteins and growth factors and were tested in an extracorporeal cellular therapy (ELAD) study to establish their effect on survival for subjects with sAH. Adults with sAH, bilirubin ≥8 mg/dL, Maddrey's discriminant function ≥ 32, and Model for End‐Stage Liver Disease (MELD) score ≤ 35 were randomized to receive standard of care (SOC) only or 3‐5 days of continuous ELAD treatment plus SOC. After a minimum follow‐up of 91 days, overall survival (OS) was assessed by using a Kaplan‐Meier survival analysis. A total of 203 subjects were enrolled (96 ELAD and 107 SOC) at 40 sites worldwide. Comparison of baseline characteristics showed no significant differences between groups and within subgroups. There was no significant difference in serious adverse events between the 2 groups. In an analysis of the intent‐to‐treat population, there was no difference in OS (51.0% versus 49.5%). The study failed its primary and secondary end point in a population with sAH and with a MELD ranging from 18 to 35 and no upper age limit. In the prespecified analysis of subjects with MELD < 28 (n = 120), ELAD was associated with a trend toward higher OS at 91 days (68.6% versus 53.6%; P = .08). Regression analysis identified high creatinine and international normalized ratio, but not bilirubin, as the MELD components predicting negative outcomes with ELAD. A new trial investigating a potential benefit of ELAD in younger subjects with sufficient renal function and less severe coagulopathy has been initiated. Liver Transplantation 24 380–393 2018 AASLD. BODY.ABBREVIATIONS: AASLDAmerican Association for the Study of Liver Diseases AHalcoholic hepatitis ALTalanine transaminase ASTaspartate transaminase CIconfidence interval CTcomputed tomography DFdiscriminant function DICdisseminated intravascular coagulation DSMBdata and safety monitoring board EASLEuropean Association for the Study of the Liver ELADextracorporeal cellular therapy HRhazard ratio IL1Rainterleukin 1 receptor antagonist INRinternational normalized ratio ITTintent‐to‐treat MELDModel for End‐Stage Liver Disease OSoverall survival PEthphosphatidyl ethanol sAHsevere alcoholic hepatitis SDstandard deviation SOCstandard of care STOPAHsteroids or pentoxifylline for alcoholic hepatitis TEAEtreatment‐emergent adverse event TESAEtreatment‐emergent serious adverse event UFultrafiltrate WBCwhite blood cell Severe alcoholic hepatitis (sAH), a syndrome which occurs in persons with heavy alcohol abuse, typically presents as liver failure with jaundice and coagulopathy.1 After initial presentation of sAH (Maddrey's discriminant function [DF] ≥ 32), mortality varies between 40% and 60% depending on the presence of secondary organ complications and treatment. The pathogenesis of liver failure in alcoholic hepatitis (AH) is complex and includes hepatic steatosis associated with inflammation, hepatocellular damage, and rapid cholestasis. Increased translocation of the gut due to excessive alcohol is thought to exacerbate inflammation in acute events, creating a vicious cycle, where inflammation, oxidative stress, and cholestatic toxin accumulation with consequent cellular damage perpetuate each other.2, 3, 4 Currently, treatment of sAH uses medical therapy with an anti‐inflammatory or immunosuppressive regimen.3 A recent study, steroids or pentoxifylline for alcoholic hepatitis (STOPAH), aimed to show the effectiveness of the current methods of treating sAH using prednisolone and/or pentoxifylline5 and showed no overall impact on 90‐day survival.55, 6, 7 Increasingly, nonresponders to medical therapy are considered to be candidates for early transplantation8, 9 because the majority of these patients will not survive a 6‐month period of alcohol abstinence required by common protocols.10 However, to narrow the use of the limited organ pool, cellular therapies have been suggested as an alternative option to current medical treatment for sAH by providing anti‐inflammatory effects and promoting hepatocellular regeneration.11, 12 The concept of liver cell therapy in sAH is based on the assumption that by providing hepatocellular support, the impaired liver cells can recover, inhibiting further degeneration and enabling recovery. A proteomic analysis of C3A cell products identified numerous proteins with anti‐inflammatory properties, such as interleukin 1 receptor antagonist (IL1Ra), the expression of which could be stimulated by various proinflammatory cytokines elevated in AH.13 Also C3A cells have been shown to express antiapoptotic and anti‐oxidative mechanisms that could further dampen the hepatocellular injury caused by inflammation and oxidative stress. In addition, C3A cells express several growth factors, such as transforming growth factor, vascular endothelial growth factor, and so on, involved in the natural regeneration naturally following the acute phase response as the first line of response to injury.14 In a prior phase 2 study, VTI‐206, C3A cellular therapy did not show survival benefit in end‐stage liver disease but a positive trend toward improved survival in a subset of sAH patients.15 The VTI‐208 study (NCT01471028) was conducted to evaluate the safety and efficacy of ELAD with respect to overall survival (OS) of subjects with sAH. BODY.PATIENTS AND METHODS: BODY.PARTICIPANTS: Subjects ≥18 years of age with a medical history of heavy alcohol abuse with a maximum of 6 weeks between the last intake of alcohol and rapid onset of jaundice (serum bilirubin ≥ 8 mg/dL) and coagulopathy (Maddrey's DF ≥ 32) were enrolled in VTI‐208. Stratum A was populated with subjects who had either liver biopsy–confirmed sAH or 2 of the following: hepatomegaly, AST > ALT, leukocytosis, or ascites. Subjects with sAH and underlying chronic liver disease other than alcoholic liver disease documented by liver biopsy, laboratory findings, and/or a medical history were randomized in a separate stratum B. Patients with end‐stage cirrhosis, as indicated by craniocaudal liver size <10 cm on ultrasound, liver volume <750 cc on computed tomography (CT) or magnetic resonance imaging, or portal vein thrombosis, were excluded. Also excluded were patients with Model for End‐Stage Liver Disease (MELD) > 35, platelets < 40,000/mm3, and individuals with severe concomitant diseases, uncontrolled bleeding, infection that was clinically unresponsive to antibiotics, hemodynamic instability, or those on chronic dialysis. BODY.STUDY DESIGN: In this open‐label trial, subjects were randomized to either receive standard of care (SOC) as defined by the American Association for the Study of Liver Disease (AASLD) and the European Association for the Study of the Liver (EASL) guidelines (control group) or SOC plus ELAD. ELAD treatment consisted of drawing blood from the subject via a dual‐lumen catheter using an extracorporeal pumping unit and then separating the plasma fluid (ultrafiltrate [UF]) from the cellular components using a specifically designed UF generator cartridge. While the cellular components are returned to the subject via the venous access, the UF is circulated at a high flow rate through 4 metabolically active hollow‐fiber ELAD cartridges containing approximately 440 g of C3A cells. After circulation through the cartridges, the UF passes through a 0.2‐µm pore‐size filter, is recombined with the cellular components of the subject's blood, and is returned to the subject through the dual‐lumen catheter (Fig. 1). For anticoagulation, site‐based protocols for continuous hemofiltration were used, with the exception of citrate. Figure 1The ELAD system is an extracorporeal human hepatic cell‐based liver treatment. During ELAD treatment, blood is drawn from the subject via a dual‐lumen catheter using an extracorporeal pumping unit, and then is separated by a specifically designed UF generator cartridge. The UF contains proteins the size of albumin and smaller but does not contain larger proteins such as antibodies. The UF is circulated at a high flow rate through the ELAD cartridges, which contain approximately 440 g of C3A cells. After circulation through the ELAD cartridges, the UF passes through a 0.2‐µm pore size cell filter, is recombined with the cellular components of the subject's blood, and is then returned to the subject via the dual‐lumen catheter. ELAD treatment was conducted continuously for 120 hours unless subjects deteriorated and became futile, withdrew consent, or subjects responded quickly after 72 hours. Subjects were followed for at least 91 days after randomization of the last subject. During that time, subjects had daily visits the first week unless discharged and then at days 7, 14, 21, 28, 63, and 91. To ensure unbiased medical management, health assessments during the follow‐up period were performed by an independent physician/nurse practitioner applying SOC. All subjects received weekly home health care service visits that included weight measurement, nutritional consultation, and a blinded phosphatidyl ethanol (PEth) to detect posttreatment alcohol use. Upon initiation of the VTI‐208 study, subjects also consented and were enrolled into an extension study, VTI‐208E to follow‐up for 5 years to assess the continued survival of both arms. The study was approved by all institutional review boards/ethics committees, and written informed consents were obtained from all participants or participants' legally authorized representative. BODY.OBJECTIVES: The primary objective of the study was to evaluate safety and efficacy of ELAD with respect to OS up to at least study day 91, with follow‐up protocol VTI‐208E providing additional survival data up to a maximum of 5 years. The secondary objectives were to evaluate the proportion of survivors at study days 28 and 91. BODY.ASSESSMENT OF EFFICACY: OS was assessed using a Kaplan‐Meier survival analysis of the intent‐to‐treat (ITT) population using a log‐rank test. BODY.ASSESSMENT OF SAFETY: Treatment‐emergent adverse events (TEAEs) and treatment‐emergent serious adverse events (TESAEs) were documented for both arms of the study population. For events occurring in ELAD subjects, the investigator assessed a potential relationship with the ELAD treatment. All TESAEs during the study were reviewed by an independent data and safety monitoring board (DSMB). BODY.LABORATORY TESTING: Standard blood testing was used to measure total bilirubin, international normalized ratio (INR), and creatinine for the calculation of MELD score. The site's certified clinical laboratories were also responsible for collecting all blood cell counts and other biochemistry data that were needed at baseline and follow‐up visits. BODY.STANDARD OF CARE: Trial sites were instructed to apply AASLD/EASL guidelines for treatment of complications of liver disease. For specific sAH SOC, concomitant medication, such as steroids, pentoxifylline, N‐acetylcysteine, and antibiotics were documented. BODY.RANDOMIZATION: Randomization was conducted using a predetermined block size (4) with a 1:1 ratio of ELAD to control subjects. Separate randomization schedules (stratified randomization) were made for strata A and B. The allocation concealment was computerized based on an electronic case report file system. Once a subject was enrolled in the study, the principal investigator received a code from the sponsor's enrollment hotline and randomization occurred online. BODY.STATISTICAL METHODS: BODY.POWER CALCULATION: On the basis of a former controlled study, 40% of control subjects and 60% of ELAD‐treated subjects were expected to survive at least until day 91.15 Under the assumption of a proportional hazards model, this leads to median survival estimates of approximately 68 days and 122 days for control and ELAD subjects, respectively. A sample size of 100 subjects per group was calculated to provide a power of at least 0.95 using a log‐rank test comparing 2 survival curves with a minimum of 91‐day follow‐up for the last subject enrolled. No interim analysis was performed. BODY.PRIMARY ANALYSIS: The OS of the ITT population was done by Kaplan‐Meier analysis using a log‐rank test to evaluate the null hypothesis of equality of survival curves with a minimum follow‐up of 91 days after the enrollment of the last subject. Model‐based estimates (and confidence limits) of median survival by treatment group and hazard rates, along with the hazard ratio (HR) and confidence interval (CI), were produced. BODY.SECONDARY ANALYSES: A chi‐square test was used to evaluate the proportion of subjects who survived at the end of study day 28 and the end of study day 91 based on the ITT population. BODY.COMPARABILITY OF GROUPS: Demographics and baseline parameters were compared with the 2 groups by exact chi‐square methods for discrete variables and parametric (t test) or nonparametric (Mann‐Whitney U) comparisons for continuous variables. The following parameters were assessed for comparability: time between alcohol abuse and hospital admission, time between hospital admission and randomization, age, sex, baseline total bilirubin, INR, creatinine, MELD, baseline status of infection and antibiotic treatment, use of vasopressors, ventilation, dialysis and steroids use as SOC. BODY.COVARIATES AND PRESPECIFIED SUBGROUP ANALYSIS: The statistical plan predefined factors related to standard demographics, selected baseline characteristics, medical history, regional and geographical site–related factors, and SOC. Comparisons between treatment groups according to these predefined criteria were carried out, analyzed, summarized, and reviewed. The following demographic and baseline characteristics were evaluated: age, sex, baseline MELD score and its components, hepatic encephalopathy grade, and baseline white blood cell (WBC) count. BODY.STUDY POPULATIONS: The ITT population included all randomized subjects assigned to the group to which they were randomized, regardless of actual treatment. The safety population was defined by actual treatment received by subjects. BODY.RESULTS: BODY.PARTICIPANTS: Between March 2013 and February 2015, 374 patients were screened at 40 sites in the United States, United Kingdom, and Australia. Of those screened, 203 subjects met the criteria for enrollment and were randomized to either ELAD (n = 96) or control (n = 107) comprising the ITT population. Also, 171 subjects did not meet 1 or more of the inclusion or exclusion criteria as detailed in the legend of Fig. 2. The uneven distribution of subjects between ELAD and control is due to the block randomization by site (blocks of 4, 2 each of ELAD and control). Because individual sites did not enroll sufficient subjects to fulfil 1 or more complete blocks, there was, by chance, an imbalance of ELAD and control subjects. Figure 2Flowchart and disposition of patients during the study until July 2015. Of the 374 subjects screened for the study, 171 did not meet inclusion criteria or presented with exclusion criteria, most frequently the inability to provide informed consent (n = 39), MELD score > 35 (n = 44), evidence of reduction in total bilirubin of 20% or more in the previous 72 hours (n = 42), and evidence of significant concomitant disease with expected life expectancy of <3 months (n = 29). *The ITT population is “as randomized.” Of the 96 subjects randomized to ELAD, 2 subjects deteriorated and became unstable before ELAD could be initiated; those subjects did not receive ELAD. One subject randomized to control received ELAD. In a separate safety analysis, the populations were analyzed “as treated.” †During the 91‐day follow‐up, 1 patient in the ELAD group and 1 in the control group were “lost to follow‐up” and 2 patients in the control group withdrew consent, so the outcome is known in 95 and 104 subjects in ELAD and control. ‡As of July 2015, using data from the VTI‐208 Extension study, the outcome is known for 94 ELAD and 102 Control subjects; 1 ELAD subject and 3 Control subjects were lost to follow‐up and 1 ELAD subject and 2 Control subjects withdrew consent. The baseline characteristics of the 203 subjects are presented in Table 1. The majority of subjects were in stratum A (n = 193), with only 10 subjects assigned to stratum B, therefore both strata were combined and further analysis will be presented for the total population (n = 203). There were 13 of 107 control and 15 of 96 ELAD subjects who were diagnosed with sAH with a confirmatory biopsy. The groups had comparable time between last alcohol intake, hospitalization, and randomization. Groups had an even distribution of sex, MELD score (mean, 27.3), bilirubin (mean, 25.1 mg/dL), and subjects who required vasopressor therapy (38 subjects), with no statistically significant differences. Ventilator support was required in 8 ELAD subjects and 3 control subjects. At baseline, 2 ELAD subjects and no control subjects were on acute dialysis. The control group had more subjects <35 years old than the ELAD group. Table 1Baseline Characteristics Prespecified Subgroups ELAD (n = 96)Prespecified Subgroups Control (n = 107)Baseline CharacteristicsELAD (n = 96)Control (n = 107)MELD < 28 (n = 51)MELD ≥ 28 (n = 45)Age < 46.9 years (n = 43)Age ≥ 46.9 years (n = 53)MELD < 28 (n = 69)MELD ≥ 28 (n = 38)Age < 46.9 years (n = 58)Age ≥ 46.9 years (n = 49)Acute AH (group A) 92 (95.8) 101 (94.4) 47 (92.2) 45 (100) 42 (97.7) 50 (94.3) 64 (92.8) 37 (97.4) 55 (94.8) 46 (93.9) AH superimposed on underlying liver disease not due to alcohol (group B) 4 (4.2) 6 (5.6) 4 (7.8) 0 (0) 1 (2.3) 3 (5.7) 5 (7.2) 1 (2.6) 3 (5.2) 3 (6.1) Days between last alcohol and hospital admission 19.8 ± 14.2 19.8 ± 16.2 17.7 ± 13.7 22.2 ± 14.5 17.8 ± 12.7 21.5 ± 15.1 16.9 ± 14.9 23.7 ± 17.7 18.9 ± 16.7 19.7 ± 15.7 Days between site admission and randomization 10 ± 6.1 9.4 ± 7.8 8.3 ± 5.8 12.0 ± 5.9 9.2 ± 5.7 10.7 ± 6.4 7.8 ± 4.9 12.3 ± 10.8 9.7 ± 9.1 9.0 ± 5.9 Age, years 46.5 ± 9.1 44.8 ± 10.7 45.6 ± 8.4 47.5 ± 9.8 38.2 ± 5.6 53.2 ± 4.7 45.8 ± 10.7 43.0 ± 10.5 36.6 ± 5.9 54.4 ± 5.8 Sex Males 55 (57.3) 65 (60.7) 25 (49.0) 30 (66.7) 22 (51.2) 33 (62.3) 40 (58.0) 25 (65.8) 33 (56.9) 32 (65.3) Females 41 (42.7) 42 (39.3) 26 (51.0) 15 (33.3) 21 (48.8) 20 (37.7) 29 (42.0) 13 (34.2) 25 (43.1) 17 (34.7) Subjects with infection and systemic antibiotics 58 (60.4) 58 (54.2) 27 (52.9) 31 (68.9) 26 (60.5) 32 (60.4) 36 (52.2) 22 (57.9) 33 (56.9) 25 (51.0) >2 episodes of dialysis in last week 2 (2.1) 0 (0) 0 (0) 2 (4.4) 0 (0) 2 (3.8) 0 (0) 0 (0) 0 (0) 0 (0) Intubated 8 (8.3) 3 (2.8) 5 (9.8) 3 (6.7) 6 (14.0) 2 (3.8) 1 (1.4) 2 (5.3) 1 (1.7) 2 (4.1) Encephalopathy grade 0 52 54 32 20 25 27 35 19 26 28 1‐2 39 41 18 21 17 22 26 15 29 12 3‐4 3 9 1 2 1 2 6 3 3 6 On vasopressors 18 (18.8) 20 (18.7) 3 (5.9) 15 (33.3) 5 (11.6) 13 (24.5) 7 (10.1) 13 (34.2) 11 (19.0) 9 (18.4) Bilirubin from baseline MELD, mg/dL 26.21 ± 9.7 24.07 ± 8.3 23.0 ± 7.9 29.8 ± 10.3 24.8 ± 8.0 27.4 ± 10.8 22.1 ± 7.8 27.7 ± 8.2 23.8 ± 9.4 24.4 ± 6.9 INR 2.0 ± 0.6 2.1 ± 0.5 1.8 ± 0.3 2.3 ± 0.6 2.0 ± 0.5 2.0 ± 0.6 1.8 ± 0.4 2.4 ± 0.5 2.2 ± 0.5 1.9 ± 0.5 Creatinine, mg/dL 1.1 ± 0.9 0.9 ± 0.5 0.7 ± 0.3 1.5 ± 1.2 0.9 ± 0.6 1.2 ± 1.1 0.8 ± 0.3 1.3 ± 0.6 0.9 ± 0.6 0.9 ± 0.4 MELD 27.6 ± 3.9 27.1 ± 3.8 24.5 ± 1.9 31.1 ± 2.4 27.0 ± 3.3 28.0 ± 4.4 24.7 ± 1.9 31.3 ± 2.3 27.8 ± 3.8 26.2 ± 3.6 Albumin, g/dL 2.8 ± 0.7 2.6 ± 0.6 2.7 ± 0.6 3.0 ± 0.7 2.8 ± 0.6 2.8 ± 0.8 2.5 ± 0.6 2.9 ± 0.7 2.7 ± 0.7 2.6 ± 0.6 AST, U/L 129.2 ± 60.9 140.9 ± 81.4 137.3 ± 64.4 120.1 ± 56.3 130.3 ± 62.2 128.2 ± 60.5 151.0 ± 83.9 122.7 ± 74.2 143.0 ± 90.7 138.5 ± 69.7 ALT, U/L 61.3 ± 44.1 61.4 ± 38.9 58.0 ± 42.5 65.1 ± 45.9 56.1 ± 44.6 65.5 ± 43.6 63.9 ± 42.8 57.0 ± 30.7 57.9 ± 30.9 65.6 ± 46.7 WBC, 109/L 15.4 ± 8.0 14.4 ± 8.1 14.1 ± 6.8 16.8 ± 9.0 15.7 ± 7.5 15.0 ± 8.4 13.3 ± 7.7 16.5 ± 8.5 14.9 ± 9.5 13.9 ± 6.2 Liver size ultrasound 19.1 ± 4.8 19.3 ± 3.9 18.6 ± 4.8 18.9 ± 4.7 20.8 ± 5.0 17.8 ± 4.2 19.5 ± 3.7 19.1 ± 4.2 19.7 ± 4.0 18.8 ± 3.8 Ascites present 55 65 25 30 31 34 39 26 37 28 On steroids 44 (45.8) 53 (49.5) 24 (47.1) 20 (44.4) 20 (46.5) 24 (45.3) 39 (56.5) 14 (36.8) 27 (46.6) 26 (53.1) On pentoxifylline 33 (34.4) 34 (31.8) 17 (33.3) 16 (35.6) 14 (32.6) 19 (35.8) 21 (30.4) 13 (34.2) 27 (46.6) 7 (14.3) NOTE: Data are given as n (%) or mean ± SD. Table 1 provides the baseline characteristics of all subjects as well as of the predefined subgroups regarding age and baseline. MELD. With the exception of a higher ratio of younger patients in the control arm, none of the baseline characteristics reached statistical difference by either unpaired comparison (t test or Mann‐Whitney U test were appropriate) or chi‐square analysis for distribution analysis. Approximately half of the subjects in both groups were on steroids and one‐third were treated with pentoxifylline at baseline. There were no observed differences in the use of steroids between groups at screening, days 1‐7, or as the study progressed. At baseline, 58 of 96 (60.4%) ELAD and 58 of 107 (54.2%) control (n.s.) subjects were on systemic antibiotics for infection. There were also no significant differences between ELAD and controls in this subpopulation with respect to disease severity based on MELD or age. The safety population was defined according to whether subjects actually received either ELAD treatment or SOC because this would best define the safety profile of ELAD treatment. This population differed from the ITT population because 2 subjects randomized to ELAD did not actually receive ELAD treatment, because they became unstable before the therapy could be initiated and 1 subject randomized to control inadvertently received ELAD treatment, leading to 95 subjects in the ELAD treatment group and 108 subjects in the control group. BODY.TREATMENT EXPOSURE: Of the 96 subjects randomized to receive ELAD treatment, 45 completed treatment of 120 hours, 37 completed between 72 and 120 hours, 12 received <72 hours of treatment, and 2 became too unstable to initiate. The control subject who received ELAD was treated for <72 hours. In total, 95 subjects were exposed to ELAD therapy, of which 13 subjects did not receive minimum treatment. There were 50 subjects who did not complete the protocol‐specified maximum treatment of 120 hours. These subjects were discontinued due to the following: clotting (n = 2), investigator deeming patient too unstable to continue (n = 9), adverse event (n = 18), futility due to bilirubin increase >25% (n = 1), subject withdrawing consent (n = 1), subject wanting to be removed from ELAD (n = 2), need for interruption to conduct CT scan or dialysis (n = 2), mechanical issue (n = 1), catheter issues (n = 3), family wanting to proceed with comfort measures only (n = 1), component issue (n = 3), and 7 subjects were discontinued without documentation that explained why. BODY.OS AND SURVIVAL PROPORTION IN THE ITT: The OS with a minimum follow‐up time of 91 days, as evaluated according to the statistical plan, was not different between the groups as depicted in Fig. 3 (HR, 1.03; 95% CI, 0.69‐1.53) with 47.9% death rate in the ELAD group and 47.7% in the control group. At the time of database lock, 51% of ELAD subjects and 49.5% of control subjects were alive (Fig. 2). Figure 3The Kaplan‐Meier analysis is given for the ITT population. At database lock, 46 subjects had died in the ELAD group and 53 in the control group. Remaining at risk were 49 in the ELAD group and 53 in the control group. The survival proportion at days 28 and 91 was comparable between the ELAD and control groups, with 76.0% versus 80.4% and 59.4% versus 61.7%, respectively. BODY.EFFECT OF ELAD TREATMENT EXPOSURE ON 91‐DAY MORTALITY: Of the 14 subjects receiving <72 hours of treatment, including 2 where treatment was not initiated due to progressive instability at the time ELAD arrived, 10 (71.4%) subjects died within 91 days. Of the 82 subjects completing the minimum treatment of 72 hours for per protocol analysis, 29 (35.4%) subjects died. Mortality differed minimally between subjects receiving between 72 and 120 hours (12/37) and completing 120 hours (17/45; 32.4% versus 37.8%). BODY.COVARIATES AND PROSPECTIVE SUBGROUP ANALYSIS: Analyses of the prespecified subgroups of baseline age and MELD greater or less than the baseline medians (46.9 years and 28, respectively) revealed that there were trends toward worse outcomes in subjects with MELD and/or age greater than the baseline medians. Conversely, subjects with MELD and/or age less than the baseline medians experienced better outcomes (Fig. 4). The majority of the study population (n = 120) presented with MELD < 28, in which ELAD (n = 51) was associated with lower mortality: 29.4% versus 43.5% in the control group (n = 69; HR, 0.58; P = 0.08). Comparison of the baseline characteristics in this subgroup revealed no significant differences between subjects randomized to ELAD versus control that could have confounded this result (Table 1). Figure 4The Kaplan‐Meier analysis is given for the prespecified subgroup analysis with respect to age and baseline MELD. (upper right) The subgroup with age < 46.9 years, at database lock, 14 subjects had died in the ELAD group and 26 in the control group. Remaining at risk were 29 in ELAD and 32 in the control group. (upper left) The subgroup with age > 47 years, at database lock, 32 subjects had died in the ELAD group and 25 in the control group. Remaining at risk were 20 in the ELAD group and 22 in the control group. (lower right) The subgroup with MELD < 28, at database lock, 15 subjects had died in the ELAD group and 30 in the control group. Remaining at risk were 35 in the ELAD group and 37 in the control group. (lower left) The subgroup with MELD > 28, at database lock, 31 subjects had died in the ELAD group and 21 in the control group. Remaining at risk were 14 in the ELAD group and 16 in the control group. Those with MELD ≥ 28 (n = 83) had a mortality rate of 68.9% in the ELAD group (n = 45) versus 55.3% in the control group (n = 38; HR, 1.50; P = 0.15). Further analysis of the components of MELD that could be responsible for this effect revealed a trend toward poor survival in subjects who received treatment with ELAD who had severe kidney damage, as defined by creatinine > 1.5 mg/dL, and serious coagulopathy, as defined by INR > 2.5. A high creatinine or high INR predicted a less favorable outcome on ELAD whereas high bilirubin showed no effect on relative survival between the groups. In the subgroup of the study population that presented with age <46.9 years (n = 101), ELAD (n = 43) was associated with lower mortality compared with the control group (n = 58): 32.6% versus 44.8% (HR, 0.63; P = 0.17). Comparison of the baseline characteristics revealed no significant differences between ELAD and control subjects that could have explained this result (Table 1). In the study population with ≥46.9 years (n = 102), there was a tendency toward higher mortality in ELAD (n = 53) versus in control (n = 49): 60.4% versus 51.0% (HR, 1.35; P = 0.26). Although not prespecified, survival in subjects with a combination of both MELD < 28 and age < 46.9 years (n = 59) was significantly better in the ELAD group (n = 26) than in the control group (n = 33; 100% versus 73%; P = 0.006) at 91 days. BODY.STANDARD OF CARE: The 33 of 96 (34.4%) ELAD subjects and 45 of 107 (42.1%) control subjects received a 7‐day standard dose >25 mg of prednisone. Documented reasons for not administering steroids, such as renal failure or infection, were more frequent (n.s.) in the ELAD group; 58 out of 96 (60.4%) ELAD subjects presented with baseline infection under systemic antibiotic therapy compared with 58 out of 107 (54.2%) control subjects. Of the subgroup with baseline infection and systemic antibiotic treatment, 30 out of 58 (51.7%) ELAD subjects and 29 out of 58 (50.0%) control subjects were alive at 91 days, indicating a higher mortality than noninfected patients among both groups. Survival analyses were run for prednisone use versus no prednisone use and for infection with antibiotic treatment versus no infection. The primary end point analyses showed no difference in outcome of the study for the overall population. There was also no correlation between pentoxifylline or N‐acetylcysteine use and outcome. Comparison of procedures to manage complications did not differ between groups. In general, there was no evidence that different administration of standard care affected the outcomes between ELAD and control in the ITT or subgroup analysis. BODY.OVERT AND COVERT ALCOHOL USE: Fewer ELAD subjects had positive postdischarge PEth than controls (28% versus 52%; P < 0.05). Of 49 subjects with a positive PEth test after discharge, only 14 (28.6%) self‐reported alcohol use (P < 0.05); 45 of 49 subjects (91.8%) with positive PEth versus 46 of 71 (64.8%) with no evidence of alcohol use were alive at 91 days (P < 0.05). Subjects with alcohol use were younger and had lower MELD, creatinine, and bilirubin levels at screening and discharge than those without (P < 0.05). BODY.BIOMARKERS: ELAD treatment resulted in a significant reduction of total bilirubin compared with controls at all time points from day 2 to day 7. Significantly more ELAD subjects (56/95, 59%) reached a bilirubin reduction over 20% compared with controls (25/108, 23%) by day 7. Lille score calculation over that period showed 74.7% were responders (Lille < 0.45) in the ELAD group versus 47% in the control group (P < 0.01). In parallel, during the 7‐day period, the alkaline phosphatase decreased in the ELAD group from 188 ± 110 to 152 ± 81 U/L, whereas it increased from 173 ± 83 to 178 ± 100 U/L in the control group. None of the other routine clinical chemistry parameters showed significant changes. Concentration of IL1Ra, a key protein involved in the orchestration of the acute phase response, rose significantly during ELAD therapy, while remaining unchanged in control subjects (Fig. 5). Figure 5The IL1Ra concentrations of a subset of ELAD (n = 14) and control patients (n = 11) is depicted as means and standard errors. The IL1Ra analysis was conducted on a subset of VTI‐ 208 subjects that met the inclusion criteria for the follow‐up study, VTL 308 (NCT02612428), which focuses on a population with lower MELD and age. Subjects were only included in this subset if they had a full sample set, which included samples from Baseline, Study Days 3, 5, and 7, as well as at least 1 sample from a follow‐up at either 14 or 28 days. Although levels were comparable at baseline, the difference between levels in ELAD versus control subjects was significant (P < 0.05, MWU‐test) at study days 3, 5, and 7 (1 week). Also, in the Wilcoxon signed rank test, IL1Ra levels were elevated compared with baseline significantly at study day 3, 5, and 7 in ELAD patients (P < 0.05), but not in controls. Concentration of alpha‐fetoprotein (a protein secreted by C3A cells) reached their peak of 884,140 ng/mL within 7 days, with a median of 200,000 ng/mL. Those levels remained above 100 ng/mL in all ELAD patients within 1 month and in 68% of patients within 2 months, but all had normalized at the end of the study. BODY.SAFETY OF ELAD: The percentage of subjects in each group of the safety population that experienced TESAEs was similar between the ELAD (76.8%) and control (69.4%) groups. The percentage of subjects who experienced certain subgroups of TESAEs is shown in Table 2. Table 2TESAEs by System Organ Class Reported by More Than 2 ELAD Subjects System Organ Class Serious Adverse Events Preferred TermSubjects Reporting at Least 1 Serious Adverse EventELAD (n = 95)Control (n = 108)Blood and lymphatic system disorders Anemia 8 (8.4) 6 (5.6) Coagulopathy 3 (3.2) 0 (0.0) General disorders and administration site conditions Multiorgan failure 7 (7.4) 10 (9.3) Hepatobiliary disorders Ascites 6 (6.3) 13 (12.0) Hepatic failure 13 (13.7) 10 (9.3) Hepatorenal syndrome 3 (3.2) 9 (8.3) Infections and infestations Sepsis 4 (4.2) 2 (1.9) Nervous system disorders Hepatic encephalopathy 8 (8.4) 6 (5.6) Renal and urinary disorders Renal failure 4 (4.2) 1 (0.9) Renal failure acute 6 (6.3) 12 (11.1) Respiratory, thoracic, and mediastinal disorders Respiratory failure 5 (5.3) 2 (1.9) Vascular disorders Gastrointestinal hemorrhage 7 (7.4) 6 (5.6) NOTE: Data are given as n (%). These data reflect the safety population (see flowchart in Fig. 2). The number of subjects experiencing system organ class–related serious events and the percentage of subjects is shown. According to Good Clinical Practice Guidelines for Safety Reporting, the determination of seriousness was based on whether the event met one of the following criteria: life threatening, lead to hospital admission and or prolonged hospitalization, upgraded the subject to intensive care, led to death or continued disability, resulted in congenital/birth defects or the event was serious in the opinion of the investigator. Reported serious events were reviewed by a blinded DSMB. In case of a disagreement with the investigator's assessment, the investigator was informed and final assessment required consensus. Deaths occurring during the 91‐day study period were also comparable between groups, with 39 deaths occurring in the ELAD group and 41 in the control group. On the basis of the study investigators' assessments, 2 deaths in the ELAD group were judged to be related to the interventional treatment: 1 was a precipitation of disseminated intravascular coagulation (DIC) that occurred in a subject with baseline MELD > The number of subjects experiencing TEAEs were balanced between the ELAD and control groups, except for anemia (44% versus 16%, respectively), thrombocytopenia (35% versus 11%), coagulopathy (31% versus 12%), and hypotension (31% versus 17%). BODY.DISCUSSION: AH is a serious condition with a high mortality and no effective treatment. This study enrolled a relatively large group of subjects with a well‐defined clinical diagnosis of AH. The mean MELD score was 27.3 in these subjects, suggesting a 3‐month mortality of approximately 50%,16 highlighting the severity of liver failure in this study. The study failed its primary and secondary end point in a population with sAH (Maddrey's DF > 32) and with a MELD ranging from 18 to 35 and no upper age limit. Prospective subgroup analysis for MELD < 28 and age less than the baseline median showed strong trends toward improved survival, whereas older patients and those with higher MELD scores had less favorable outcomes on ELAD. Subanalysis of MELD components suggests that more severe coagulopathy with INR > 2.5 reduced the tolerance of extracorporeal treatment. Although C3A cells have been shown to produce clotting factors in vitro and ex vivo, those effects seem not to outweigh the complex derangements of coagulation observed in advanced liver failure, which are not only an expression of reduced synthesis, but also of increased consumption due to DIC, frequently precipitated by looming infection. The inability to use citrate for anticoagulation with the ELAD system—due to its negative effect on the C3A cells—may have contributed to this problem because heparin has been discussed to be an anticoagulant of second choice in extracorporeal liver support in patients prone to bleeding.17 However, a recent publication showed that low‐dose heparin was associated with an acceptable benefit/risk profile when used for extracorporeal liver support even in severely ill patients with severe acute liver failure.18 Outcomes were worse for subjects treated with ELAD whose MELD scores were ≥28 due to elevated creatinine indicating that kidney failure increases the risk while being treated with ELAD. ELAD cannot remove toxins by filtration or dialysis and thus does not provide renal support. Because of the lack of this function, potential benefits of ELAD cannot compensate for the usual adverse effects of extracorporeal therapy, such as contact activation and anticoagulation, once a subject is in renal failure. Interestingly, the beneficial effects seen in subjects with MELD scores below the baseline median of 28 seemed to manifest themselves not at the time of treatment, but within the following 2‐3 weeks (Fig. 4). This suggests the primary mechanism of action of C3A cellular therapy is not secondary organ failure support such as in extracorporeal albumin dialysis, but in aiding in recovery of the liver, which is known to take 2‐4 weeks. This is also supported by the fact that age was a second strong covariate that determined response to ELAD and liver regeneration is well‐known to decrease with age. One hypothesized mechanism for how C3A cells may aid in recovery is based on C3A cells supporting the defective acute phase response in vivo. This is exemplified by the significant IL1Ra elevation in the ELAD group, which was not seen in controls. The reduction of bilirubin in the ELAD group exceeds what can be explained by the dilution in the extracorporeal circuit, and furthermore, C3A cells in ELAD have no mechanism to drain bilirubin into the bile. The parallel reduction of alkaline phosphatase in the ELAD group versus an increase in the control group may suggest bile flow can improve while on ELAD. Although the response measured by the Lille score, the most validated measure for therapy response in AH,19 was significantly better in ELAD, it did not translate into a successful OS benefit, which the authors interpret as a consequence of the worsening of the outcome in nonresponders in the ELAD arm. Similarly, patients with high MELD scores and of older age had worse outcomes, diluting a potential survival benefit indicated by more Lille responders. Although the results of subgroup analyses must be interpreted with caution, the subgroups according to MELD, creatinine, and coagulation were in fact prespecified, interrelated, and seen in large groups, and the authors applied accepted guidelines in reporting subgroup analyses.20 The subgroup analysis related to age alone was also prespecified. However, because the combined subgroup analysis with respect to age and MELD had not been prespecified, the authors take the results of this combined analysis with specific caution. Confirming the diagnosis of AH with liver biopsy in this patient population is not routinely performed in many centers. Both AASLD guidelines21 for management of AH as well as recommendations from the National Institute on Alcohol Abuse and Alcoholism1 consider using a liver biopsy for AH diagnosis but recognize the barriers in acquiring a biopsy in this patient population. Our study did not require liver biopsy for diagnosis, and it followed the clinical diagnostic criteria put forth by these groups. It is possible that patients have other underlying diagnoses, but we would not expect the distribution between the treatment and control groups to be different. Only in 16 and 13 patients in the ELAD and the control groups, respectively, did the investigator perform a biopsy to confirm the clinical diagnosis. The teroids or Pentoxifylline for Alcoholic Hepatitis (STOPAH) trial showed no benefit in 90‐day survival in steroid‐treated patients. Thus, the findings of our study gain increasing importance because there is an unmet need for effective therapy for this highly prevalent, highly mortal hepatic disorder. Because there were limitations in the study design associated with the patient heterogeneity and a potential uncertainty of diagnosis in 10%‐20% of subjects, a confirmatory study is underway to investigate the reproducibility of these observations through the conduct of a prospective, randomized, controlled clinical study in patients with lower MELD and age (VTL 308‐NCT02612428). If successful, younger patients with AH who do not respond to medical therapy without renal failure or severe coagulopathy could undergo cellular therapy. This would allow the donor organ pool considered for early transplantation in AH to be directed to patients with more advanced coagulopathy or renal failure.

TITLE: Incontinence and Nocturia in Older Adults After Hip Fracture: Analysis of a Secondary Outcome for a Parallel Group, Randomized Controlled Trial Objective: To test the effect of a follow-up clinic on urinary incontinence (UI) and nocturia among older adults with hip fracture. Method: Fifty-three older adults (≥65 years) 3 to 12 months following hip fracture were enrolled and randomized to receive usual care plus the intervention (B4), or usual care (UC) only. The B4 group received management by health professionals, with need-based referrals. UI, nocturia, and quality of life were measured with questionnaires at baseline, 6 months, and 12 months. Results: There were 48 participants included in this analysis, and at baseline, 44% of study participants self-reported UI. At final assessment, six out of 24 B4 participants and 12 out of 24 UC participants reported UI. Four out of five study participants reported nocturia at baseline; this did not decrease during the study. Discussion: Following hip fracture, many older adults report UI and most report nocturia. Health professionals should be aware of the high occurrence of urinary symptoms among older adults post hip fracture. BODY.INTRODUCTION: Urinary incontinence (UI) and nocturia are significant health problems for older adults; the consequences of which can have far-reaching impact on the health system. Whereas UI is “the complaint of any involuntary loss of urine” (Abrams et al., 2010, p. 213), nocturia is the need to wake during sleep to void (Bosch & Weiss, 2010). The prevalence of urinary dysfunction varies by gender and residential setting. For UI, the frequency ranges from 17% to 55% and 11% to 34% among community-dwelling older women and men, respectively (Thom, 1998), and is higher among residents in long-term care (Aggazzotti et al., 2000). Nocturia increases with age, with prevalence ranging from 74% to 77% among older women and 69% to 93% for older men (Bosch & Weiss, 2010). Waking more than twice/night is associated with lower health-related quality of life (QoL; Tikkinen et al., 2010). In the United States, the estimated direct annual associated costs are US$16.3 billion (Wilson, Brown, Shin, Luc, & Subak, 2001) and US$1.5 billion (Holm-Larsen, 2014) for UI and nocturia, respectively. Many of these costs result from falls and fractures, as both incontinence and nocturia increase risk of these injuries (Brown et al., 2000). Specifically, UI (Johansson, Hellstrom, Ekelund, & Milsom, 1996) and nocturia (Asplund, 2006) are risk factors for hip fracture. Overall control of continence is an interplay between the urethra and the detrusor muscle of the bladder; the pathophysiology of urinary dysfunction can be neurogenic (Ginsberg, 2013) and/or myogenic (Brading, 1997) in origin. Neurogenic urinary dysfunction is commonly a result of damage or disease of the central nervous system that leads to inability of the urethral sphincter to adjust when bladder pressure increases (Ginsberg, 2013). In contrast, myogenic urinary dysfunction results from structural changes to the detrusor smooth muscle leading to increased nerve activity and bladder overactivity (Brading, 1997). Following surgery, there may be increased risk of urinary dysfunction, possibly due to indwelling catheters (Sorbye & Grue, 2013), but there are other likely causes such as increasing age, health status, and other surgical-related factors (Chiarelli, Byles, Parkinson, & Gibson, 2006; Palmer, Baumgarten, Langenberg, & Carson, 2002). Urinary dysfunction may persist beyond the post-acute period (Chiarelli et al., 2006), placing individuals at risk of further fall-related injury (Chiarelli, Mackenzie, & Osmotherly, 2009), sleep fragmentation (Asplund, 2004), and reduced QoL (Dubeau, Simon, & Morris, 2006). Despite this, UI and nocturia are frequently underinvestigated and managed (Edwards et al., 2011). Contributing to the low management rates is the associated stigma that creates barriers for older adults to seek medical help for their symptoms (Wang et al., 2014). This gap in assessment and reluctance to discuss urinary problems can lead to social isolation (Yip et al., 2013), and instigate or amplify a spiral of mobility loss, with consequences for QoL. In this study, the assessment of urinary dysfunction was included as part of a comprehensive geriatric assessment to improve mobility among community-dwelling older adults after hip fracture (Cook et al., 2011). Specifically, this article reports the results from the evaluation and management of UI and nocturia as part of a geriatric clinic for older adults after hip fracture. It also describes the associations between UI, nocturia, and QoL. BODY.METHOD: BODY.PARTICIPANTS: This was an analysis of a secondary outcome from a randomized controlled trial testing a follow-up clinic for older adults after hip fracture in Vancouver, Canada (Cook et al., 2011). The study included 53 community-dwelling men and women aged 65 years or older from Metro Vancouver with a recent hip fracture (3-12 months). The exclusion criteria included older adults who, prior to the fracture, were unable to walk 10 m, diagnosed with any type of dementia, and/or older adults who upon hospital discharge moved to a residential care facility. Approval to conduct the study was obtained from the university and local hospital institutional review boards. Study participants provided written informed consent prior to taking part in the study. BODY.RECRUITMENT, RANDOMIZATION, AND MEASUREMENT TIME POINTS: Clinicians from three collaborating teaching hospitals assisted with participant recruitment. After enrollment into the study, a registered physiotherapist assessed participants, and then the research coordinator used a web-based system to determine participant group allocation. Randomization was stratified by gender and hospital site, and an independent academic statistical company generated the sequence for randomization to maintain allocation concealment and reduce the risk of bias. The physiotherapist also assessed outcomes again at 6 and 12 months. BODY.INTERVENTION: The full protocol for this study and a description of the intervention is provided elsewhere (Cook et al., 2011). In summary, participants randomized to the intervention (B4) were provided usual care (UC) and a comprehensive geriatric assessment (Panel et al., 2011). The focus was on key elements deemed important and relevant to mobility recovery after hip fracture including, bone health, balance, cognition, and continence. Following the assessment by the geriatrician, participants were offered personalized clinical management strategies. One area for intervention addressed concerns with UI and/or nocturia. If appropriate, participants were referred to a continence nurse for management. Control participants followed the UC pathway including follow-up with their surgeon and family physician, and were offered the follow-up clinic at the end of their participation in the study. BODY.CONTINENCE AND NOCTURIA QUESTIONNAIRE: Participants were asked questions related to UI (Huang et al., 2007) and nocturia (Vaughan et al., 2010) based on previous literature. The questions (and possible responses) for continence are located in Table 1. Table 1.UC and Nocturia Questions Asked of Participants at Three Times Points Over the 12 Months (Baseline, 6 Months, and 12 Months). QuestionResponsesUC (Huang et al., 2007) 1. During the last 12 months, have you ever leaked urine or lost control of your urine? YesNo 2. How often does this leakage of urine usually occur? Daily ≥1 time/week (but not every day) ≥1 time/month (but not every week) <1 time/month Do not know 3. Does this leakage interfere with your activities? YesNo Nocturia (Vaughan et al., 2010) 4. During the night, how many times do you have to get up to urinate, on average? None 1 2 3 4+ 5. How much of a problem is this for you? Not a problem A bit of a problem Quite a problem A serious problem Note. UC = urinary continence. BODY.QOL: The ICEpop CAPability measure for Older people (ICECAP-O; Coast et al., 2008), a capability and well-being measure developed for use with older adults, was used to capture perceptions of QoL. BODY.DESCRIPTIVE MEASURES: Date of birth, gender, number of chronic conditions, and time since fracture were recorded. Height and weight was measured; body mass index (BMI) was calculated as weight (kg)/height (m2). BODY.STATISTICAL ANALYSIS: Participant characteristics were described using mean (standard deviation) or median (interquartile range [IQR]) if data were skewed; categorical and ordinal data were reported as counts and proportions. Point biserial correlations were used to explore the relation between presence of UI and nocturia and overall QoL (ICECAP-O index score) at the three time points. In addition, one-sided Fisher’s exact test was used to explore associations with QoL and the degree of UI frequency and severity, and the number of nightly nocturia occurrences, using Stata v13 (StataCorp, 2015). BODY.RESULTS: Fifty-three participants were recruited to the study: 26 participants were randomized to the B4 group and 27 participants to UC group (Figure 1). During the study, one B4 participant was lost to follow-up and one UC participant discontinued participation before the final assessment time point. There were missing data from one B4 participant and two UC participants: therefore, there were 24 participants in each group who contributed data to these analyses. Figure 1.Flow diagram for the B4 study continence variables. Study participants had an average (SD) age of 79.6 (7.9) years (range = 65-98 years). There were 17 out of 48 (35%) men, they had a median (IQR) BMI of 25.5 (4.8) and were diagnosed with a median (IQR) of 3 (3.5) chronic conditions. At baseline, 21 out of 48 (43.7%) of all study participants reported UI, including nine out of 24 (37.5%) from the B4 group and 12 out of 24 (50%) from the UC group. There were five intervention participants who reported UI at baseline, but did not report UI at midpoint or final assessment. At final assessment, there were four B4 participants with two new cases for a total of six B4 participants who reported UI. There were 10 out of 12 UC participants (who reported UI at baseline) with two new cases, for a total of 12 participants. In the UC group, there were three participants who reported UI at baseline and final assessment, but did not report UI at midpoint. Most study participants reported nocturia at baseline; the number and relative percentage of participants reporting nocturia were similar between the groups and did not change over the course of the study. Based on a review of B4 participants’ charts, four B4 participants were offered a referral to a continence nurse. Table 2 provides a summary of variables of interest across three time points. Table 2.Continence Outcomes by Group at Three Time Points. BaselineMidpointFinalUCB4UCB4UCB4UI  Number of participants (%) 12 (50.0) 9 (37.5) 7 (29.2) 4 (16.7) 12 (50.0) 6 (25.0)  Frequency (%)   Daily 1 (4.2) 0 (0.0) 3 (12.5) 0 (0.0) 3 (12.5) 2 (8.3)   ≥1 time/week (but not every day) 4 (16.7) 1 (4.2) 1 (4.2) 4 (16.7) 2 (8.3) 0 (0.0)   ≥1 time/month (but not every week) 1 (4.2) 3 (12.5) 1 (4.2) 0 (0.0) 4 (16.7) 2 (8.3)   <1 time/month 5 (20.8) 3 (12.5) 2 (8.3) 0 (0.0) 3 (12.5) 2 (8.3)   Do not know 1 (4.2) 2 (8.3) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)  Interferes with activities (%) 0 (0.0) 1 (4.2) 1 (4.2) 2 (8.3) 2 (8.3) 2 (8.3) Nocturia  Number of participants (%) 18 (75.0) 20 (83.3) 19 (79.2) 20 (83.3) 19 (79.2) 21 (87.5)   Once/night 13 (54.2) 11 (45.8) 13 (54.2) 10 (41.7) 11 (45.8) 11 (45.8)   Two or more times/night 5 (20.8) 9 (37.5) 6 (25.0) 10 (41.7) 8 (33.3) 10 (41.7)  Severity of problem (%)   Not a problem 11 (45.8) 15 (65.4) 14 (58.3) 16 (66.7) 11 (45.8) 17 (70.8)   Bit of a problem 5 (20.8) 5 (20.8) 2 (8.3) 3 (12.5) 6 (25.0 2 (8.3   Quite a problem 1 (4.2) 0 (0.0) 3 (12.5) 0 (0.0) 1 (4.2) 2 (8.3)   A serious problem 1 (4.2) 0 (0.0) 0 (0.0) 1 (4.2) 1 (4.2) 0 (0.0)   No response 6 (25.0) 4 (16.7) 5 (20.8) 4 (16.7) 5 (20.8) 3 (12.5) Note. There were 48 participants who contributed data to these analyses, including 24 participants in each group (control and intervention groups). UC = usual care; UI = urinary incontinence. Using a one-sided Fisher’s exact test, no statistically significant differences between groups for UI at midpoint (.125, CI = [0.00, 0.360], p = .247) or final assessment (.25, CI = [0.00, 0.515], p = .068) were found. There were no statistically significant differences between groups for the presence of nocturia at midpoint (.042, CI = [0.00, 0.262], p = .500) or final assessment (.083, CI = [0.00, 0.293], p = .350). A statistically significant association between the presence of UI and QoL (r = −.36; p = .016) was found only at midpoint. Negative coefficients were interpreted as participants who did not experience the presence of UI and had higher QoL (ICECAP-O) scores. There were no significant associations between nocturia and QoL (Table 3). Table 3.Associations Between Presence of Urinary Incontinence and Nocturia With Quality of Life Using Point Biserial Correlation Coefficients at Three Time Points, Regardless of Group Allocation. Coefficient p UI and QoL  Baseline (n = 47) −.10 .51  Midpoint (n = 44) −.36 .016  Final (n = 48) −.21 .15 Nocturia and QoL  Baseline (n = 47) −.03 .83  Midpoint (n = 44) .24 .11  Final (n = 48) .03 .84 Note. UI = urinary incontinence; QoL = quality of life. BODY.DISCUSSION: The consequences of UI and nocturia can be life changing, and pose negative consequences on physical, social, and emotional well-being (Ramage-Morin & Gilmour, 2013). Older adults with UI are more susceptible to health problems such as falls (Chiarelli et al., 2009), fractures (Asplund, 2006; Brown et al., 2000; Johansson et al., 1996), and may have higher rates of admission to a residential care facility (Thom, Haan, & Van Den Eeden, 1997). At baseline, more than two of every five participants reported UI, and most participants experienced nocturia to varying degrees. A significant statistical association between UI and lower QoL was noted at midpoint. However, there was no difference between groups for urinary-related impairments; this may be due to the small sample size, the intervention, and/or the stigma associated with this health concern, which may have led some participants to underreport their symptoms Stigma associated with UI (Wang et al., 2014) might prevent older adults from seeking help and taking part in out-of-home activities, leading to increased risk of social isolation, loneliness, and decreased independence (Ramage-Morin & Gilmour, 2013). Many older adults believe that UI and nocturia are a normal part of aging and, therefore, avoid discussing the topic with their doctor (Umlauf, Goode, & Burgio, 1996). Thus, UI is frequently underreported. Despite the fact that UI and nocturia can affect older adults’ health, QoL, and well-being, there have been few studies that focused on addressing UI-related health concerns after hip fracture. Current evidence suggests a number of ways to manage UI and nocturia including lifestyle advice, physical therapy and pelvic muscle strengthening (Baigis-Smith, Smith, Rose, & Newman, 1989), biofeedback, scheduled voiding, behavioral therapies, medication, and surgical procedures (Abrams et al., 2010). Studies have found that physical therapy and behavioral therapy are effective treatments for UI and nocturia for older adults (Aslan, Komurcu, Beji, & Yalcin, 2008). Study participants in the B4 group were assessed by a physiotherapist who prescribed individualized balance and strength exercises to improve balance, gait, and vestibular function. A recent systematic review noted that a nonspecific exercise program does not change UI symptoms (Bo & Herbert, 2013), which perhaps speaks to exercise specificity and requires that pelvic floor exercises be included within an overall balance and strength routine. Targeted exercise may have benefits beyond addressing bladder retraining. For example, engaging in a regular physical activity program that includes balance and core strengthening could also provide additional safeguards for fall prevention. In addition, studies on healthy community-dwelling older adults found that maintaining an active lifestyle can reduce the risk of developing UI (Bo & Herbert, 2013). For nocturia, there has been limited investigation; however, a small study (n = 30 men) noted a decrease in symptoms and better sleep associated with a walking program (Sugaya et al., 2007). There are limitations noted with this study. First, this was a small study and the results may not be generalizable to all older adults with hip fracture. Second, participants were community dwelling, which do not represent the full spectrum of older adults with hip fracture. Third, the study was underpowered to detect any changes in UI and nocturia between groups. Other limitations include using a self-report measure to detect participants’ impairments in urinary function and not enquiring about urinary function prior to the fracture. Finally, the study did not document individual recommendations to participants or measure study participant uptake of recommendations from the clinic intervention outside of clinician visit frequency and type. Despite these limitations, the importance of this work is that it highlights the moderate to high prevalence of UI and nocturia in a group of older adults after hip fracture—already a vulnerable group at greater risk of future injuries. These issues must be addressed, including minimizing associated stigma because of the increased risk of falls and fractures, social isolation, and potential institutionalization that can ensue if continence is not managed. Identifying and managing UI and nocturia (and their consequences) early are key to mitigating health risks, reducing risk of falls and fractures, and potentially improving QoL. As this can be a sensitive topic for some people, strategies are required to address psychosocial concerns for urological health in older adults to make management more approachable for this and other at-risk populations.

TITLE: Opioid sparing effect of low dose ketamine in patients with intravenous patient-controlled analgesia using fentanyl after lumbar spinal fusion surgery ABSTRACT.BACKGROUND: The opioid sparing effect of low dose ketamine is influenced by bolus dose, infusion rate, duration of infusion, and differences in the intensity of postoperative pain. In this study, we investigated the opioid sparing effect of low dose ketamine in patients with intravenous patient-controlled analgesia (PCA) using fentanyl after lumbar spinal fusion surgery, which can cause severe postoperative pain. ABSTRACT.METHODS: Sixty patients scheduled for elective lumbar spinal fusion surgery were randomly assigned to receive one of three study medications (K1 group: ketamine infusion of 1 µg/kg/min following bolus 0.5 mg/kg, K2 group: ketamine infusion of 2 µg/kg/min following bolus 0.5 mg/kg, Control group: saline infusion following bolus of saline). Continuous infusion of ketamine began before skin incision intraoperatively, and continued until 48 h postoperatively. For postoperative pain control, patients were administered fentanyl using IV-PCA (bolus dose 15 µg of fentanyl, lockout interval of 5 min, no basal infusion). For 48 h postoperatively, the total amount of fentanyl consumption, postoperative pain score, adverse effects and patients' satisfaction were evaluated. ABSTRACT.RESULTS: The total amount of fentanyl consumption was significantly lower in the K2 group (474 µg) compared to the control group (826 µg) and the K1 group (756 µg) during the 48 h after surgery. Pain scores at rest or with movement, the incidence of adverse events and patient satisfaction were not significantly different among the groups. ABSTRACT.CONCLUSIONS: Low-dose ketamine at 2 µg/kg/min following bolus 0.5 mg/kg significantly reduced the total amount of fentanyl consumption during the 48 h after lumbar spinal fusion surgery without increasing adverse effects. BODY.INTRODUCTION: Patient-controlled analgesia (PCA) with opioids has been widely used for the control of post-operative pain. However, due to adverse effects such as nausea and vomiting, the adjuvant use of variable drugs has emerged [1]. Among these, ketamine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, has been known to improve postoperative opioid effectiveness [2]. It has opioid sparing effect, inhibits windup and central sensitization, and reduces the development of chronic pain [3-5]. Low-dose ketamine is defined as a bolus dose of less than 2 mg/kg when given intramuscularly or less than 1 mg/kg when administered via the intravenous (IV) or epidural route. In continuous IV administration, low-dose ketamine is defined as a rate of ≤ 20 µg/kg/min [6]. However, the effect of low-dose ketamine is controversial [7-10]. It is thought that the method and dose of ketamine administration, and differences in the intensity of postoperative pain, may affect the results [11]. In this study, we investigated low-dose ketamine in terms of its opioid sparing effect in patients with IV PCA using fentanyl after lumbar spinal fusion surgery, which can cause severe postoperative pain. BODY.MATERIALS AND METHODS: Approval was obtained from the Institutional Review Board before study commencement. After receiving written informed consent, 60 healthy patients with an American Society of Anesthesiologists physical status classification of I-II, aged between 28 and 70 years old, and who were scheduled for elective major lumbar spinal surgery were enrolled in this randomized, placebo-controlled, double-blinded study. The type of surgery was posterior decompression and posterior lumbar interbody fusion with instrumentation. The exclusion criteria comprised pregnancy, psychiatric problems, chronic alcoholism, drug abuse, inability to use PCA, and lack of communication ability. Patients were randomly assigned to one of three groups: 1) K1 group, ketamine infusion of 1 µg/kg/min following bolus 0.5 mg/kg; 2) K2 group, ketamine infusion of 2 µg/kg/min following bolus 0.5 mg/kg of ketamine and 3) Control group, identical volume of normal saline infusion following bolus of normal saline before surgical incision. Continuous IV infusion of ketamine or saline following bolus dose started before skin incision intraoperatively, and continued until 48 h postoperatively via infusor (Autoinfusor®, Ace Medical Co, Seoul, Korea), which included the relevant study medication diluted with saline. All patients and the investigators collecting the postoperative data were blinded to the randomization. A standardized anesthesia regimen was followed. All patients received glycopyrrolate 0.2 mg IM for premedication 30 min before surgery. Anesthesia was induced with propofol 2 mg/kg, remifentanil infusion 20 µg/min, and rocuronium 0.6 mg/kg. Anesthesia was maintained with desflurane, nitrous oxide (50%) and remifentanil infusion 5-20 µg/min. Approximate ten minutes before the end of surgery, a 50-100 µg bolus dose of fentanyl and 30 mg ketorolac were given intravenously. Ondansetron 8 mg was IV injected simultaneously and an additional 8 mg was mixed to PCA to prevent postoperative nausea and vomiting. After skin closure, desflurane and remifentanil were discontinued, and residual neuromuscular block was reversed with pyridostigmine and glycopyrrolate. The trachea was extubated when patients responded to verbal commands, and recovered spontaneous respiration. For postoperative pain control, patients were administered fentanyl using IV-PCA (bolus dose 15 µg of fentanyl, lockout interval of 5 min, no basal infusion). After surgery, patients were observed in the postanesthetic care unit for 1 h before transferal to the ward. PCA fentanyl use was evaluated for 48 h after surgery. The pain was evaluated with a 100-mm visual analog scale (VAS) (0 = no pain; 100 = worst imaginable pain). Patients were asked to evaluate their maximal degree of pain. Pain scores were recorded at rest and with movement at 1, 6, 24, and 48 h after surgery. Pain with movement was defined as pain on rolling, sitting, or coughing. Adverse events such as sedation, nausea, vomiting, headache and psychomimetic symptoms (vivid dreams or hallucination) were assessed. When moderate or severe nausea or vomiting was present, patients were administered metoclopramide 10 mg or ondansetron 4 mg. Patients were also asked to rate their overall satisfaction with the PCA experience on a five-point scale (very satisfied, satisfied, neutral, dissatisfied, very dissatisfied) [12] at 48 h after operation. The primary outcome measure of this study was the total amount of fentanyl consumption during the 48 h after operation, while the secondary outcome measures were the pain scores, adverse effects, and patient satisfaction. Sample size was predetermined using a power analysis to achieve 90% chance (β = 0.1) with an assumed significance level of α = 0.05. From pilot study, the calculated minimum sample size was 17 patients in each group. A larger number of patients were included to allow for possible incomplete data collection or patient dropout. Data are presented as mean ± SD, or number of patients. The statistical analysis was performed using SPSS for Windows (version 14, SPSS Inc., Chicago, IL, USA). A one-way analysis of variance was used to compare the continuous variables among the groups. If a significant difference was noted, Bonferroni multiple comparison test was used to determine intergroup differences. Categorical variables were analyzed using the chi-square test or Fisher exact test, as appropriate. A P value of less than 0.05 was considered statistically significant. BODY.RESULTS: Of the 60 original patients, eight were lost from the study. Five patients stopped PCA use early because of severe postoperative nausea and vomiting (two patients in control, one patient in K1, two patients in K2), or because of PCA use error in three patients (one patient in control, one patient in K1, one patient in K2). There were no significant differences among the three groups with respect to demographic data, duration of operation and anesthesia, or the total amount of remifentanil used intraoperatively (Table 1). The total amount of fentanyl consumption was significantly lower in the K2 group (474 µg) compared to the control group (826 µg) and the K1 group (756 µg) during the 48 h after surgery (P < 0.05) (Table 2). VAS scores for pain at rest or with movement at 1, 6, 24 and 48 h postoperatively were similar among the three groups (Table 2). No patient experienced bad dreams or hallucinations. The incidence of adverse events such as nausea, vomiting, dizziness, headache, and sedation were comparable among the groups (Table 3). The patient satisfaction rate was similar among the three groups (Table 4). BODY.DISCUSSION: Ketamine has been used as a general anesthetic and analgesic for various pain conditions over the past several decades. Since Foster and Fagg reported the discovery of the NMDA receptor in 1987, ketamine has been used as a potential anti-hyperalgesic agent given its actions as a non-competitive NMDA-receptor antagonist. However, it remains a controversial drug due to undesirable adverse effects. It is clear that a distinction must be made between high-dose ketamine as an anesthetic agent and low-dose ketamine as an anti-hyperalgesic agent. There may even be a third dose range in which ketamine has no analgesic potency on its own but when used in combination with an opioid, yields an opioid sparing effect and superior pain relief than would occur for either drug alone [6]. There is some indication that ketamine in a dose of 0.3 mg/kg does not interfere with µ-opiate receptors as, in this dose, its analgesic effects cannot be antagonized with the µ-opiate antagonist naloxone [13]. Only interference with NMDA receptors may be involved in ketamine analgesia at this dose [14], suggesting that 0.3 mg/kg ketamine may be "selective" for NMDA receptors [15]. Furthermore, Tucker et al. [16] reported that low steady doses of ketamine (serum ketamine 30-120 ng/ml) could be combined with µ opioid agonists to improve their analgesic effect without adverse effects in the clinical setting. In addition, for a ketamine infusion rate of 1-6 µg/kg/min in combination with a loading dose, there is evidence of anti-hyperalgesic, analgesic and opioid sparing effects [17,18]. We therefore investigated a loading dose of 0.5 mg/kg, followed by 1 or 2 µg/kg/min, as in these earlier studies. In this study, the total amount of fentanyl used until 48 hours post-surgery was significantly lower in those patients who received ketamine infusion of 2 µg/kg/min following 0.5 mg/kg bolus dose compared to the those who received saline or ketamine infusion of 1 µg/kg/min following 0.5 mg/kg bolus dose. This result is consistent with other studies that used a similar dose [19-22]. In contrast with this result, however, Jaksch et al. [23] reported that ketamine 2 µg/kg/min following 0.5 mg/kg bolus dose had no effect on postoperative pain reduction in patients after knee arthroscopic surgery, but this study differed from our own in that ketamine was infused for two hours post-surgery, whereas we infused for 48 hours. Considering the short half-life of ketamine, the duration of infusion as well as the optimal dose is an important component of the opioid sparing effect. Zakine et al. [24] compared ketamine infusion during only the intraoperative period with that for the perioperative period (intraoperative plus postoperative 48 h). They demonstrated that low dose ketamine improved postoperative analgesia with a significant decrease of morphine consumption when its administration was continued until 48 h postoperatively. Another result from the current study is that there was no difference in fentanyl consumption between the K1 group (ketamine infusion of 1 µg/kg/min following 0.5 mg/kg bolus dose) and the control group. Yamauchi et al. [25] reported that continuous low-dose ketamine improved analgesic effects after cervical spine surgery but not after lumbar surgery, and indicated that the ketamine dose required for the opioid sparing effect varies by the intensity of pain. Accordingly we investigated patients who underwent posterior lumbar interbody fusion with instrumentation, which has the capacity to cause similar severe pain. We also did not use basal infusion in PCA to prevent the masking effect of ketamine from pain reduction by continuous opioid infusion. In this study, subjects did not report any psychomimetic effects such as bad dreams and hallucinations. There were no advantages observed in terms of pain VAS and a reduction in adverse effects despite lower opioid consumption in those patients who received a ketamine infusion of 2 µg/kg/min following 0.5 mg/kg bolus dose. As such, the opioid-sparing effect of ketamine in the clinical setting may be questionable, but may still be useful for patients who need a high dose of opioids or are unusually sensitive to opioids [11]. The central limitation of this study is that we could not use higher doses of ketamine in light of potential psychomimetic effects. Other studies have used a similar dose without reporting adverse psychiatric effects, and Schmid et al. [6] reported that the incidence of psychomimetic effects and cognitive impairment was negligible at dose less than 2.5 µg/kg/min IV and increased with higher doses. .The purpose of this study was to find the minimum effective dose of ketamine and as such further studies that assess what represents a clinically effective dose of ketamine may be needed. In conclusion, low-dose ketamine of 2 µg/kg/min following bolus 0.5 mg/kg, but not not 1 µg/kg/min following bolus 0.5 mg/kg, significantly reduced the total amount of fentanyl consumption during 48 h after lumbar spinal fusion surgery without increasing the incidence of side effects.

TITLE: Closed-Loop Insulin Delivery Using a Subcutaneous Glucose Sensor and Intraperitoneal Insulin DeliveryFeasibility study testing a new model for the artificial pancreas ABSTRACT.OBJECTIVE: Attempts to build an artificial pancreas by using subcutaneous insulin delivery from a portable pump guided by an subcutaneous glucose sensor have encountered delays and variability of insulin absorption. We tested closed-loop intraperitoneal insulin infusion from an implanted pump driven by an subcutaneous glucose sensor via a proportional-integral-derivative (PID) algorithm. ABSTRACT.RESEARCH DESIGN AND METHODS: Two-day closed-loop therapy (except for a 15-min premeal manual bolus) was compared with a 1-day control phase with intraperitoneal open-loop insulin delivery, according to randomized order, in a hospital setting in eight type 1 diabetic patients treated by implanted pumps. The percentage of time spent with blood glucose in the 4.4–6.6 mmol/l range was the primary end point. ABSTRACT.RESULTS: During the closed-loop phases, the mean ± SEM percentage of time spent with blood glucose in the 4.4–6.6 mmol/l range was significantly higher (39.1 ± 4.5 vs. 27.7 ± 6.2%, P = 0.05), and overall dispersion of blood glucose values was reduced among patients. Better closed-loop glucose control came from the time periods excluding the two early postprandial hours with a higher percentage of time in the 4.4–6.6 mmol/l range (46.3 ± 5.3 vs. 28.6 ± 7.4, P = 0.025) and lower mean blood glucose levels (6.9 ± 0.3 vs. 7.9 ± 0.6 mmol/l, P = 0.036). Time spent with blood glucose <3.3 mmol/l was low and similar for both investigational phases. ABSTRACT.CONCLUSIONS: Our results demonstrate the feasibility of intraperitoneal insulin delivery for an artificial β-cell and support the need for further study. Moreover, according to a semiautomated mode, the features of the premeal bolus in terms of timing and amount warrant further research. In patients with type 1 diabetes, the near-normal glucose control required to prevent long-term complications (1,2) remains difficult to achieve (3). Indeed, the incidence of hypoglycemia increases when glucose control approaches normal glucose levels (4). For this reason the development of an “artificial pancreas” has been a goal for >30 years (5,6). An artificial β-cell requires three elements: a continuous insulin delivery device, a continuous glucose monitoring system, and a control algorithm linking insulin delivery to glucose measurements (3,7,8). The recent development of better performing continuous glucose sensors renewed the potential feasibility of closed-loop insulin delivery (9–11). Short-term initiatives in the clinical research setting were reported in recent years but showed some limitations (12–14). Key limiting factors were, first, delays in the modulation of insulin action related to subcutaneous infusion and, second, time lags in glucose detection due to either the placement of the sensors in the interstitial compartment of subcutaneous tissue or the internal structure of implanted intravenous sensors (15). To reduce glucose deviations at mealtimes, a hybrid option of closed-loop insulin delivery includes a manual priming bolus (16). Reported benefits of intraperitoneal insulin infusion from implantable pumps include fast insulin action and low basal plasma insulin levels, resulting in tight glucose control and a low incidence of hypoglycemic events (17). The feasibility of automated closed-loop insulin delivery from implantable pumps has been demonstrated in clinical trials performed with the Long-Term Sensor System, which coupled these devices with an intravenous glucose sensor (18). Our approach to optimize closed-loop glucose control includes the use of closer to physiological intraperitoneal insulin delivery, subcutaneous glucose sensing, and a proportional-integral-derivative (PID) algorithm with a manual premeal bolus, resulting in a hybrid PID (HyPID) system. The objective of this study was to test the feasibility of such an approach. We investigated patients in the same controlled hospital setting while testing the HyPID system and when following their usual self-management. This approach marks a difference from the previously reported closed-loop trials, which considered home-use periods for comparison with in-clinic closed-loop studies (13,16). BODY.RESEARCH DESIGN AND METHODS: Eight patients with type 1 diabetes, treated by an implanted pump using intraperitoneal delivery (model MMT-2007D; Medtronic Diabetes, Northridge, CA) and infusing U-400 regular insulin (Insuplant; sanofi-aventis, Paris, France) for at least 3 months, were enrolled. Inclusion criteria were the following: age 18–70 years, insulin delivery within 15% of expected accuracy for the 60 days preceding the trial, plasma anti-insulin antibody level <30% according to a radioimmunoassay of free and total anti-insulin antibody using a technique adapted from that of Palmer et al. (19), written informed consent, and health insurance coverage by the French Social Security System. Exclusion criteria were pregnancy, breast feeding, plasma creatinine >150 μmol/l, serum alanine aminotransferase and aspartate aminotransferase above twice the highest limit of the normal range, total blood Hb <12 g/dl, any cardiovascular event during the last 6 months, any evolving ischemic or proliferative diabetic retinopathy on eye fundus examination for the previous year, and any known or suspected allergy to glucose sensor components. The study protocol was approved by the regional ethics committee Comité de Protection des Personnes Sud Mediterranée IV, Montpellier, France, on 11 September 2007. The study was authorized by Agence Française de Sécurité Sanitaire des Produits de Santé on 29 November 2007 and registered under the reference number 2007-A00696-47 (www.afssaps.sante.fr). Subjects were admitted to the hospital for a total of 86 h, which was divided into a preparation phase (14 h), a control (open-loop) phase (24 h), and a closed-loop phase (48 h). The order of the control and closed-loop phases was randomized. At admission (day 1, 1800), two subcutaneous glucose sensors (Medtronic Diabetes), similar to those used in Medtronic's CGMS and Guardian RT systems, were inserted in the abdominal area and were calibrated against a capillary blood glucose (CBG) value 2 h after insertion and then every 4 h. The second sensor was used as a backup in case the first sensor failed to track glucose. At 2000, patients were instructed to program their insulin bolus for dinner and to remain fasting until the following morning. On day 2, 20 min before the 800 experiment start, an intravenous catheter was placed in an antecubital vein for frequent blood sampling. Blood samples were then drawn (for later blood glucose and plasma insulin measurements) every 20 min at the start of each meal for a period of 2 h (800–1000, 1300–1500, and 1900–2100, considered as “early postprandial periods” for breakfast including 40 g carbohydrate and lunch and dinner both including 70 g carbohydrate), every hour from 800 to 2200 except for early postprandial periods, and every 2 h from 2200 to 800 (considered as “nonpostprandial periods”). During the 24-h control phase, the patients were instructed to monitor their diabetes by seven CBG tests performed before and 2 h after each meal and at bedtime and to program their pump according to the self-monitoring data. Sensor glucose data were monitored in real time and were patient blinded. Table 1Glucose control and plasma insulin levels during closed-loop and control phases in eight type 1 diabetic patients treated by implanted insulin pumps and monitored by a subcutaneous glucose sensor Whole periodNonpostprandial periodEarly postprandial periodControlClosed-loop95% CIPControlClosed-loop95% CIPControlClosed-loop95% CIPBlood glucose (mmol/l) 7.6 ± 0.7 7.4 ± 0.4 −1.1 to 0.5 0.401 7.9 ± 0.6 6.9 ± 0.3 −1.9 to 0.0 0.036 8.6 ± 0.9 8.8 ± 0.5 −0.9 to 1.4 0.484 Plasma insulin (mIU/l) 20.9 ± 3.5 17.7 ± 2.2 −7.9 to 1.4 0.161 11.8 ± 2.0 15.6 ± 2.1 0.9 to 6.7 0.012 30.4 ± 5.4 21.8 ± 2.7 −16.5 to −0.6 0.050 Time spent with blood glucose (mmol/l)     >10% 21.0 ± 8.0 13.6 ± 3.3 −19.0 to 4.2 0.263 18.0 ± 7.3 9.0 ± 2.6 −21.1 to 3.2 0.128 29.9 ± 11.5 27.4 ± 6.8 −20.2 to 15.2 0.866     >6.6% 57.0 ± 8.1 51.0 ± 4.9 −16.2 to 4.1 0.161 53.8 ± 8.4 42.0 ± 6.0 −22.2 to −1.4 0.036 66.5 ± 11.9 77.7 ± 4.2 −10.7 to 33.0 0.575     ≤4.4 and ≥6.6% 27.7 ± 6.2 39.1 ± 4.5 1.9 to 20.8 0.05 28.6 ± 7.4 46.3 ± 5.3 4.9 to 30.4 0.025 25.0 ± 7.0 17.7 ± 3.4 −18.7 to 4.1 0.123     <4.4% 15.3 ± 4.6 9.9 ± 2.7 −18.2 to 7.6 0.401 17.6 ± 5.4 11.7 ± 3.5 −21.9 to 10.2 0.779 8.5 ± 5.5 4.6 ± 1.4 −15.2 to 7.4 0.753     <3.3% 0.6 ± 0.5 1.6 ± 1.2 −2.3 to 4.3 0.686 0.6 ± 0.6 1.8 ± 1.4 −2.8 to 5.0 0.593 0.5 ± 0.5 1.0 ± 0.7 −1.8 to 2.8 0.593 Data are arithmetic means ± SEM and 95% CI. During the 48-h closed-loop phase, the pump's insulin infusion rate was automatically modulated according to the algorithm. Sensor glucose data and insulin delivery rate were monitored in real time and were patient blinded. Fifteen minutes before meals, a manually programmed insulin bolus, consisting of 30% of the amount the patient would have programmed according to premeal blood glucose levels and meal carbohydrate content, was delivered. For safety purposes, CBG tests were also performed every hour from 800 to 2200 and every 2 hours from 2200 to 800. In addition, each time the sensor glucose value decreased to <4.4 mmol/l (80 mg/dl) or increased to >13.2 mmol/l (240 mg/dl) and when patients reported symptoms of suspected hypoglycemia, a CBG test was performed. It should be noted that procedures to respond to hypoglycemia and sustained hyperglycemia were also followed during the control phase, although at home the subject might not have such close monitoring. BODY.SYSTEM CONSIDERATIONS: The closed-loop system is made up of three components: a subcutaneous glucose sensor, the insulin delivery algorithm (running on a laptop computer), and the intraperitoneal insulin infusion pump. The computer receives sensor data using a radiofrequency protocol and sends commands to the pump using the Bluetooth protocol. A modified personal pump communicator, set up to receive commands from a Bluetooth adapter, was used instead of the personal pump communicator used by the patient. The pump was then set to the minimum allowed basal infusion rate of 0.2 unit/h, with the algorithm delivering discrete 0.2-unit boluses as calculated based on real-time sensor glucose measurements. The mathematical algorithm used to calculate the insulin delivery rate is based on a model of the multiphasic insulin response of a β-cell (20). The first version of this algorithm was described in Steil et al. (13). The version used in this study further incorporates the effect of insulin-inhibiting insulin secretion (i.e., insulin feedback) (21). The equations describing the model are where u(n) is the insulin infusion rate calculated at time step n (which is every 1 min), and the notation (n −1) denotes the previous time step. The algorithm is tuned with the parameters KP, τI, τD, and γ and with α1, α2, and Kcl corresponding to the intraperitoneal insulin absorption kinetics. The term γIP corresponds to the inhibition by plasma insulin concentration on the delivery of insulin. Because the pump can only deliver insulin as single 0.2-unit boluses, the amount actually delivered by the pump (IB) is used to calculate the estimated plasma insulin concentration. The tuning parameters are individualized for each subject as a function of his or her total daily insulin dose. The target glucose level used for the algorithm was 100 mg/dl (5.5 mmol/l). BODY.LABORATORY MEASUREMENTS: Plasma glucose concentrations were measured by hexokinase assay (Olympus, Rungis, France). CBG measurements were performed using OneTouch Ultra meters and strips (LifeScan, Milpitas, CA). Plasma insulin was measured by a specific insulin assay (bi-insulin immunoradiometric assay; Schering CIS bio international, Gif sur Yvette, France). BODY.ASSESSMENT OF GLUCOSE CONTROL: The primary end point was the percentage of time spent with blood glucose in the 4.4–6.6 mmol/l range. All analyses were done by using the laboratory blood glucose measurements unless otherwise noted. Secondary end points included the same index for the early postprandial periods and for the nonpostprandial periods; mean blood glucose for the overall experiment, for the early postprandial periods, and for the nonpostprandial periods; and percentage of time spent with blood glucose <3.3 mmol/l and >10 mmol/l. BODY.STATISTICAL ANALYSIS: Results are expressed as arithmetic means ± SEM or SD when specified and 95% CIs of differences. Means were compared using the Wilcoxon signed-rank test. The level of significance was set at P < 0.05. Calculations and statistical analysis were performed using SYSTAT 10 (SPSS, Chicago, IL). BODY.RESULTS: Eight patients (seven male and one female) were enrolled. Patient characteristics were as follows (mean ± SD): age 59.8 ± 8.7 years, BMI 26.4 ± 3.4 kg/m2, diabetes duration 31.7 ± 15.1 years, treatment duration by implanted pump 8.5 ± 7.4 years, A1C 6.8 ± 1.0%, and daily insulin requirement 0.60 ± 0.21 units · kg–1 · day–1. BODY.SENSOR ACCURACY: Mean and median relative absolute differences (± SD) between paired sensor and laboratory blood glucose values were 15.9 ± 3.8 and 13.9 ± 2.9%, respectively, which are consistent with previous reports (16). BODY.INSULIN DELIVERY AND ALGORITHM ASSESSMENT: The correlation coefficient (R2) between the measured and algorithm-estimated plasma-insulin levels was 0.730 ± 0.067. Data for each patient are shown in Fig. 1. In general, although the magnitude of the estimated levels is higher than that of the measured levels, the kinetics observed are well described by the model. The observed difference in the slope of the estimates versus the predictions may be due to specific aspects of intraperitoneal insulin infusion. Figure 1Correlations between measured (lab) and algorithm-estimated (model) plasma insulin levels during the closed-loop phases in each of the eight type 1 diabetic patients investigated by the HyPID system. BODY.GLUCOSE CONTROL DURING CONTROL AND CLOSED-LOOP PERIODS: The distribution of blood glucose values and the mean blood glucose and plasma insulin levels are presented in Table 1. A significantly higher percentage of time was spent between 4.4 and 6.6 mmol/l during closed-loop versus control phases (39.1 ± 4.5 vs. 27.7 ± 6.2%, P = 0.05), although mean blood glucose shows no significant difference. No carryover phenomenon was detected. Tighter control was obtained for closed-loop phases in nonpostprandial periods, in which both mean blood glucose level and percentage of time spent with blood glucose between 4.4 and 6.6 mmol/l are significantly better. In contrast, early postprandial glucose control was similar during closed-loop and control periods. Of note, plasma insulin levels were significantly higher in nonpostprandial periods but significantly lower in early postprandial periods during closed-loop phases. In the nighttime period between 2200 and 800, mean blood glucose levels and percentage of time spent between 4.4 and 6.6 mmol/l were similar (Fig. 2A). A trend to better control was observed only from 2200 to 200 during closed-loop phases (6.2 ± 0.4 vs. 7.9 ± 0.9 mmol/l, P = 0.069). Data analysis in early postprandial periods showed similar glucose peak levels and time-to-glucose peak (Fig. 2B). However, for closed-loop phases, plasma insulin peak levels were significantly lower (29.7 ± 2.9 vs. 51.5 ± 8.4 mIU/l, P = 0.017) and the time to the plasma insulin peak was longer (79.9 ± 7.2 vs. 38.3 ± 7.2 min, P = 0.012). These differences were observed similarly with all three main meals (data not shown). Figure 2Blood glucose levels (mean 95% CI) during closed-loop (continuous lines) and control (dashed lines) phases in the eight type 1 diabetic patients investigated by the HyPID system. A: from 2200 to 0800. B: From 1 h before to 5 h after meal start. Figure 3 indicates the higher mean frequency of blood glucose between 4.4 and 6.6 mmol/l during closed-loop phases. It also shows a tighter interpatient distribution of glucose values in these phases. Figure 3Cumulative distribution of blood glucose values during closed-loop and control (open-loop) phases in the eight type 1 diabetic patients investigated by the HyPID system. Individual data are presented as thin continuous lines during closed-loop phases and as thin dashed lines during control (open-loop) phases. Thick lines indicate the median cohort distributions of blood glucose values. Vertical red dotted lines denote the glucose range between 4.4 (80) and 6.6 (120) mmol/l (mg/dl). In terms of safety, 13 glucose deviations <4.4 mmol/l were detected by the patients through suggestive symptoms and/or identified early by the glucose sensors during closed-loop phases, and 3 were detected during control phases. All events occurred in nonpostprandial periods. Of note, after oral glucose administration (10 g on average), a trend for earlier correction of blood glucose was observed during closed-loop phases (82.67 ± 0.81 vs. 70.33 ± 0.19 mg/dl after 20 min). BODY.CONCLUSIONS: The improved glucose control obtained during the closed-loop phases represents a valuable improvement for patients whose glucose was already well controlled as indicated by their initial A1C levels of 6.8 ± 1.0%, because it was achieved with minimal patient interaction with the system. The assessment of closed-loop effectiveness, measured by percentage of time spent in the tight near-normal glucose range, illustrates the usefulness of sensor- and algorithm-driven insulin infusion. Because hyperglycemic excursions have been associated with oxidative stress (22) and hypoglycemic deviations impair quality of life and can promote hypoglycemia unawareness, leading to the occurrence of severe hypoglycemia (23), an important goal for a closed-loop system is to reduce glucose deviations. In addition, reduced interpatient variability of glucose levels also merits notice. This result is valuable in terms of reproducibility and safety of the algorithm. The main benefit on glucose control during closed-loop phases was observed in time periods excluding the early postprandial (2-h) periods. Improvement of glucose control during these periods appears to be driven mainly by higher plasma insulin levels obtained by algorithm-driven insulin delivery. The trend to a quicker return to premeal glucose levels in the late postprandial periods, i.e., >2 h after meals, can be highlighted. However, despite the manual delivery of a premeal bolus during closed-loop phases, the early postprandial period remains a challenging situation that also was not solved in previously reported closed-loop experiments (14,15). Programming a manual bolus before meals did not seem to be as effective in our study as in a recent trial using continuous subcutaneous insulin infusion (16). Of note, in our experiments, postprandial insulin peaks were lower and later during closed-loop versus control phases. Reproduction of the dynamics of the physiological first phase of insulin secretion would require reaching higher acute plasma insulin levels corresponding both to the “cephalic phase” of insulin secretion and to the “incretin-promoted” component (24,25). Future clinical trials should evaluate the amount and timing of the manual premeal bolus to better mimic physiology. From an algorithmic consideration, we can speculate that insulin action resulting from the premeal bolus may mask the appearance of glucose, therefore delaying the increase in the insulin delivery rate by the algorithm. Blood glucose could be maintained between 3.85 and 10 mmol/l for 85% of the time in a recently reported 24-h closed-loop trial performed on eight adolescent type 1 diabetic patients in a hospital setting by combining subcutaneous insulin infusion, subcutaneous sensing, and a PID algorithm, the two latter elements being very similar to ours, except that insulin feedback was included in our algorithm (16). Glucose control was maintained in the same range only 58% of the time in the open-loop phase performed in the home environment. Our data show that blood glucose was kept between 4.4 and 10 mmol/l for 76.5% of the time in 48-h closed-loop phases, which was also significantly better than in the open-loop phases during which glucose was kept in the same range for 63.7% of the time. The evaluation of the open-loop period in the same hospital setting has, however, a stronger value for comparison. The large between-patient variability of glucose control, during performance of the closed-loop trial using subcutaneous insulin delivery reported by the authors, may represent a significant difference with our data obtained by intraperitoneal infusion (16). This difference in terms of blood glucose variability may be partially due to differences between these two routes of insulin delivery, which has already been reported in previous studies assessing implantable insulin pumps (17). Hypoglycemia is a worrisome situation in closed-loop insulin delivery. In our study, hypoglycemic deviations occurred in a limited percentage of time, which was not significantly different from that in the control phase. This observation argues for the safety of the algorithm used. Moreover, because all hypoglycemic events were either detected by the patients from suggestive symptoms and/or immediately identified by the sensor, we may expect that a low-glucose warning system based on the sensor signal would be sufficient to prevent severe hypoglycemia in patients using the closed-loop algorithm at home. In summary, our study demonstrates the feasibility, safety, and benefits on glucose control of a new alternative for closed-loop control. The reduced between-patient variability in glucose control is also worth noting. Although currently limited to a few patients in whom subcutaneous insulin delivery was considered unreliable, implanted devices for intraperitoneal insulin infusion have been shown to provide additional benefits in terms of quality of life (17). Further development is needed to improve early postprandial glucose control, requiring premeal manual intervention for bolus programming in agreement with previous trials (12,16).

TITLE: Atrial natriuretic peptide infusion and nitric oxide inhalation in patients with acute respiratory distress syndrome ABSTRACT.AIM:: To study the effects of infusion of atrial natriuretic peptide (ANP) versus the inhalation of nitric oxide (NO) in patients with an early acute respiratory distress syndrome (ARDS). ABSTRACT.METHODS:: Ten patients with severe ARDS were studied in a crossover study design, within 72 hours after starting mechanical ventilation. We studied the effects of ANP infusion (10 ng/kg/min for 1 hour) and of inhalation of NO (20 ppm for 1 hour) on hemodynamic and respiratory patient parameters, as well as the effects on plasma levels of ANP, guanosine 3',5'-cyclic monophosphate, nitrate and endothelin-1. ABSTRACT.RESULTS:: Despite an approximate 50% increase in mixed venous ANP plasma concentration (from 86 ± 21 to 123 ± 33 ng/l, P < 0.05) during ANP infusion, there were no changes in mean pulmonary artery pressure, pulmonary vascular resistance index, extravascular lung water index, or in pulmonary gas exchange. NO inhalation, in contrast, lowered mean pulmonary artery pressure (from 26 ± 1.9 to 23.9 ± 1.7 mmHg, P < 0.01), pulmonary vascular resistance index (from 314 ± 37 to 273 ± 32 dynes/cm5/m2, P < 0.05) and central venous pressure (from 8.2 ± 1.2 to 7.3 ± 1.1 mmHg, P < 0.02). Furthermore, NO inhalation improved pulmonary gas exchange, reflected by a decrease in alveolar-arterial oxygen gradient (from 41.9 ± 3.9 to 40.4 ± 3.6 kPa, P < 0.05), a small increase in oxygenation (PaO2/FiO2 from 17.7 ± 1.4 to 19.7 ± 1.1 kPa, P = 0.07) and a small decrease in venous admixture (Qs/Qt from 35.7 ± 2.0 to 32.8 ± 2.7%, P = 0.11). ABSTRACT.CONCLUSION:: This study shows that, in contrast to NO inhalation, infusion of ANP neither improves oxygenation nor attenuates pulmonary hypertension or pulmonary edema in patients with severe ARDS. BODY.INTRODUCTION: ANP, a peptide mainly secreted in the right atrium, is an important regulator of the sodium and volume homeostasis [1]. Right atrial stretch is the main trigger for the production of ANP. Apart from its natriuretic properties, ANP has vasodilating effects caused by binding to biologically active 'B receptors' [2]. Binding to these B receptors leads to activation of the enzyme particulate guanylate cyclase, which in turn enhances intracellular production of guanosine 3',5'-cyclic monophosphate (cGMP). The cGMP leads to relaxation of smooth muscle cells. ANP may, in this way, be an important regulator of pulmonary vascular tone as the lung possesses abundant binding receptors for ANP. Other important triggers for the production of ANP are hypoxia and pulmonary vasoconstriction [3, 4, 5]. Finally, another feature of ANP is in vitro improvement of the barrier function of pulmonary endothelial cells [6]. These properties suggest that ANP is an attractive agent in the treatment of patients with hypoxia and pulmonary vasoconstriction. ANP infusion improved pulmonary gas exchange under experimental hypoxic conditions in men in a recent study [7]. On the contrary, ANP infusion lowered pulmonary artery pressure but did not improve oxygenation in patients with chronic obstructive pulmonary diseases [8]. In accordance with other vasodilators, the venous admixture was even enhanced in these patients. ARDS is the extreme clinical example of pulmonary edema and hypoxia induced pulmonary vasoconstriction. Based on our findings in experimental hypoxia, we hypothesized that ANP infusion could be beneficial in ARDS patients. The promising effects of NO inhalation in ARDS [9, 10] also supported this hypothesis, because both NO and ANP act through activation of guanylate cyclase. Moreover, in a recent study, ANP infusion was shown to improve oxygenation and to lower pulmonary artery pressure in a hydrochloric acid lung injury model in pigs [11]. In the present paper, we have studied the effects of ANP infusion and NO inhalation in patients with severe ARDS in the early stage of the disease, in a nonblinded crossover design. BODY.MATERIALS AND METHODS: BODY.SUBJECTS: Ten patients with severe ARDS were enrolled between October 1994 and January 1996. Only patients with a Lung Injury Score >2.5 were included [12]. To be eligible for the study, the duration of mechanical ventilation for ARDS had to be less than 72 hours. Patients had to be in a stable hemodynamic condition. Patients were excluded if they were younger than age15, were pregnant, had a known allergy for iodine, or had a known stenosis in a femoral artery. The Local Ethics Committee approved the study protocol, and each patient's next of kin gave informed consent. BODY.MEASUREMENTS: All patients underwent right-sided heart catheterization with placement of a pulmonary artery catheter (7.5-F Swan-Ganz catheter, Model VS1721; Ohmeda, Swindon, UK). A 4-F fiberoptic catheter (Pulsiocath PV2024; Pulsion, Munich, Germany) was placed in the descending aorta through a 6-F introducer sheath (Model 616150A; Ohmeda) in one of the femoral arteries. Mean arterial pressure was recorded via the side port of the introducer sheath. The pulmonary artery catheter was used for measurements of central venous pressure, mean pulmonary artery pressure, and pulmonary artery wedge pressure, with the midchest level as zero reference. Thermodilution cardiac output (CO) was measured with injection of 10 cm3 ice-cold saline at random during the respiratory cycle. The mean value of three consecutive measurements was used for analysis. Heart rate was recorded continuously with one of the standard leads of the electrocardiogram. Cardiac index (CI), stroke index, systemic vascular resistance index and pulmonary vascular resistance index were calculated according to standard formulae. Measurements of extravascular lung water index (EVLWI), intrathoracic blood volume index (ITBVI), pulmonary blood volume index (PBVI), and right ventricular end-diastolic volume index (RVEDVI) were obtained with the thermal-dye dilution technique (COLD Z-021 system; Pulsion). Measurements were carried out with injection of 10 cm3 ice-cold indocyanin green solution (2 mg/ml). The mean value of two measurements was used for analysis. Refer to the literature for details concerning the thermal-dye dilution technique [13, 14]. Briefly, the method uses two indicators: ice-cold water and indocyanin green (ICG). Cold distributes to both intravascular and extravascular volumes, whereas ICG remains intravascular. Both indicators are injected into the right atrium, and concentration changes in time are recorded in the descending aorta. A dilution curve can thus be constructed for both indicators. CO is determined from the thermodilution curve. A mean transit time (MTT) and a downslope time (DST) can be derived from each indicator's dilution curve. The MTT is composed of the appearance time ATi, which is the time until the first indicator particle has arrived at the point of detection, and the mean time difference between the occurrence of the first particle and all the following particles. The product of CO and MTT is the volume between the site of injection and the site of detection. The DST can be derived from the extrapolated descending limb of a logarithmic transformation of the dilution curve. In a series of mixing chambers, the product of CO and DST represents the volume of the largest mixing chamber in the chain. The volumes already mentioned can be measured using the following formulae. ITTVI (ml/m2) = CI × MTTaorta(temperature) ITBVI (ml/m2) = CI × MTTaorta(ICG) PBVI (ml/m2) = CI × DSTaorta(ICG) EVLWI (ml/kg) = ITTVI - ITBVI where ITTVI represents the intrathoracic thermal volume index. Because we used the COLD system together with a Swan-Ganz catheter, we were also able to determine RVEDVI by the application of the following formula: RVEDVI (ml/m2) = CI × DSTpulmonary artery(temperature) Together with each hemodynamic measurement, arterial and mixed venous blood samples were drawn for determination of hemoglobin and blood gas analysis (Ciba-Cormig 288 blood gas analyzer; Ciba-Cormig, Medfield, MA, USA). Furthermore, arterial and mixed venous ethylenediamine tetraacetic acid blood samples were taken for determination of plasma levels of ANP, cGMP, nitrate, and endothelin-1. These samples were immediately placed on ice and were centrifuged at 3000 rpm and 0°C, for 10 min. The collected plasma samples were then stored at -70°C until final analysis. ANP was determined with a sensitive radioimmunoassay, using an immunoextraction with a C-terminal-specific antiserum (Incstar, Stillwater, MN, USA), as described previously [15]. ANP levels vary widely in normal subjects. Generally accepted normal values are 10-70 ng/l in healthy men younger than 65 years [1]. The cGMP was measured with a direct radioimmunoassay (Immuno Biological Laboratories, Hamburg, Germany) according to the method of Steiner et al [16]. Normal venous concentrations are 1-6 nmol/l. Nitrate was determined as described previously, the nitrate levels in healthy subjects with no dietary restrictions being 33 ± 3 μmol/l [17]. BODY.MECHANICAL VENTILATION AND NO ADMINISTRATION: All patients were ventilated with a Bear 1000 ventilator (Bourns Medical Systems Inc., Riverside, CA, USA) in either a volume-controlled or a pressure-controlled mode, with plateau pressures not exceeding 30 cmH2O. Positive end-expiratory pressure was guided by the lower inflection point of the pressure-volume curve. Patients were sedated with midazolam and morphine in such a way that they were unable to trigger the ventilator. When necessary, they were also paralysed with pancuronium. The ventilator settings were unchanged during the experiments. NO (600 ± 30 ppm NO in N2; Air Products, Waddinxveen, The Netherlands) was added with a continuous flow to the inspiratory limb of the ventilator circuit [18]. Flow was titrated with a high precision flowmeter (Sho-Rate™ Model 1355; Brooks Instrument B.V., Veenendaal, The Netherlands). Inspiratory concentrations of NO and NO2 were measured between the 'Y' piece of the ventilator and the endotracheal tube, using electrochemical sensors (NOx-box; Bedfont Scientific Ltd., Upchurch, Kent, UK). The sensors are calibrated every 6 months using a gas mixture containing 55 ppm NO and 5.5 ppm NO2. The limit of detection is 0.1 ppm for NO and 0.1 ppm for NO2. The range of detection is 0-200 ppm for both gases. Additional hydrophobic filters were used to protect the sensors from condensed water vapor. The inspired NO2 concentration was not allowed to exceed 1 ppm. BODY.STUDY PROTOCOL: The effects of ANP infusion and NO inhalation were studied in a crossover design. An entrance number was assigned to each patient before the start of the protocol, according to which patients started with either ANP infusion (odd numbers) or NO inhalation (even numbers). At the start of the study protocol, two baseline measurements were performed in each patient, 30 min apart. Patients then started with either human ANP infusion (Atriobiss; Clinalfa AG, Läufelfingen, Germany) at a rate of 10 ng/kg/min for 1 hour or NO inhalation of 20 ppm for 1 hour, with a third measurement at the end of this hour. The dosage regimens were based on effective and non-toxic dosages in earlier reports [7, 9, 19, 20]. A fourth measurement was carried out 1 hour after cessation of the first treatment modality. Patients then started with their second treatment modality and a fifth measurement was obtained after 1 hour of treatment. Finally, a sixth measurement was taken 1 hour after cessation of the second treatment modality. No adjustments in inotropic or ventilatory therapy were made during the study protocol. BODY.STATISTICAL ANALYSIS: Data are expressed as mean values ± SEM, unless noted otherwise. The mean value of baseline measurements 1 and 2 was used for analysis. Treatment effects were analysed with the Student t test for paired samples. When the data did not have a normal distribution, the Wilcoxon signed-rank test was used. P < 0.05 was considered to be statistically significant. Statistical analysis was performed with Excel (Version 5.0 for Windows, Microsoft Corporation, Redmond, WA, USA) and SPSS (Version 6.0 for Windows, SPSS Inc, Chicago, IL, USA). BODY.RESULTS: Patient characteristics are presented in Table 1. The mean age was 50.2 years (range, 30-66 years). Duration of mechanical ventilation before starting the study protocol was 26 hours (range, 6-60 hours). The mean Lung Injury Score was 2.8 (range, 2.5-3.25). All patients were hemodynamically stable throughout the study. There were no significant differences in the distinguished baseline measurements (measurements 1, 2, 4 and 6). The values for baseline presented in Tables 2 and 3 are the mean values of measurements 1 and 2. Table 2 presents the various variables measured during ANP infusion. Both the mixed venous and the arterial concentration of ANP increased during infusion of ANP. Statistical significance was only noted for the change in the mixed venous concentration. The same results were obtained for cGMP concentrations. None of the other parameters changed during ANP infusion. Table 3 presents the parameters measured during NO inhalation. Mean pulmonary artery pressure, pulmonary vascular resistance index and central venous pressure decreased during NO inhalation. In contrast to treatment with ANP, both arterial and mixed venous concentrations of cGMP increased significantly during NO inhalation. EVLWI did not change either during ANP infusion or during NO inhalation. In patients who responded to NO inhalation with an increase in PaO2/FiO2 >10%, EVLWI tended to be lower than in patients who did not show such a response (16.2 ± 1.7 versus 20.6 ± 3.5 ml/kg, P = 0.29). The alveolar-arterial oxygen gradient decreased significantly during NO inhalation. The venous admixture also decreased and PaO2/FiO2 increased, but neither change reached statistical significance (P = 0.11 and P = 0.07, respectively). BODY.DISCUSSION: The present study shows that ANP infusion at 10 ng/kg/min during 1 hour in patients with severe ARDS in the early stage of their disease did not reduce pulmonary hypertension, nor did it improve pulmonary gas exchange. ANP also did not influence the amount of extravascular lung water. In contrast, 20 ppm NO inhalation lowered the pulmonary artery pressure and improved pulmonary gas exchange. In a previous study, normal subjects were exposed to hypoxia by decreasing barometric pressure, and the effects of ANP infusion were monitored. An improvement in arterial oxygen saturation and a decrease in the alveolar-arterial oxygen difference were noticed under these circumstances [7]. It was then hypothesized that ANP decreased transcapillary filtration in the pulmonary circulation, thereby preventing the development of pulmonary edema under hypoxic conditions and thus improving pulmonary gas exchange. Because extravascular lung water was not measured, it was not possible to prove this hypothesis. Andrivet et al studied the effects of ANP infusion in patients with severe chronic obstructive pulmonary diseases. They found a deterioration in the ventilation-perfusion relationship during ANP infusion, with a decrease in arterial oxygenation that was masked by an increased minute ventilation [8]. The present study was designed to elucidate the effects of ANP infusion in ARDS patients during the early phase of the disease. We did not detect a measurable clinical effect of ANP infusion in these patients. Several explanations are possible for this finding. There was a significant increase in mixed venous concentration of ANP, but only a modest increase in the arterial concentration during the infusion of ANP. Since ANP was infused on the venous side of the circulation, these findings suggest that most of the infused ANP was cleared by the lung. The pulmonary circulation expresses two types of receptors for ANP: the biologically active 'B receptor', and the 'C receptor', which is reported to have a mere clearance function without biological effects [2, 21]. We found increased mixed venous concentrations of ANP without measurable clinical effects. This might suggest that the infused ANP was preferentially bound to C receptors. The increase in mixed venous cGMP concentrations indicates that the infused ANP did induce biological effects, although this was not translated into clinical signs. Another explanation for our results may be that ANP did not produce clinical effects because there was already a maximal vasodilating effect of cGMP at baseline, as the baseline plasma concentrations of cGMP were high in comparison with normal subjects. This hypothesis is refuted by the fact that NO inhalation produced clinical effects with the same baseline plasma levels of cGMP. The increase of cGMP was higher after NO inhalation than after ANP infusion, suggesting that the administered dose of ANP may have been too low. Indeed, we cannot completely exclude too low a dosage. Unfortunately, we did not measure diuresis during ANP; an increase in diuresis might have suggested an adequate dosage. On the contrary, the clinical effects in experimental hypoxia, as seen previously, were achieved with similar doses of ANP. The different administration routes of ANP and NO may explain the different effects. Pulmonary vascular resistance and pressure are mainly determined by the small resistance vessels. NO is administered near the resistance vessels of the pulmonary circulation and acts primarily on the capillary-venous compartment, as was recently shown [22]. ANP enters the pulmonary circulation on the arterial side and may bind to receptors at that site. It is imaginable, in this way, that ANP never reaches the 'target vessels', particularly since there is extensive microthrombosis in the capillaries in ARDS [23]. It is also possible that plasma levels of cGMP do not reflect local concentrations. The different clinical effects of NO inhalation and ANP infusion may thus be the result of essential different local cGMP levels in the lung. The results of the present study do not correspond with the results of ANP infusion in an experimental animal model of lung injury [11]. This indicates that the pathological substrate of ARDS may differ from the experimental models that were used. The shortcomings of experimental models to completely mimic the complex mechanisms involved in ARDS were recently reviewed in extension [24]. Generally, the clinical effects of NO inhalation in our study are in accordance with earlier reports. We did not find a significant improvement of PaO2/FiO2 (P = 0.07), possibly because of the small sample size of the study. Furthermore, it is known that approximately 65% of ARDS patients respond to inhaled NO [10]. In our study group, 50% of the patients showed an improvement in PaO2/FiO2 of more than 10%. Finally, we did not construct individual dose-response curves. It is known that patients may vary widely in optimal doses of inhaled NO. Some of our patients may therefore have received a dose that was above their optimum, which is known to deteriorate oxygenation again [25]. Furthermore, NO was administered in a continuous flow to the inspiratory limb of the ventilator circuit. It is now known that this mode of administration with noncontinuous flow modes of mechanical ventilation may produce highly variable and unpredictable concentrations of inhaled NO [26]. This may also have influenced our results in an unknown direction. EVLWI tended to decrease during NO inhalation, somewhat more in responders than in nonresponders, although not significantly. This is in accordance with the recently elucidated primary effect of NO on the venous compartment of the pulmonary capillary circulation [22]. In conclusion, this study shows that short-term ANP infusion in early ARDS does not improve pulmonary gas exchange or pulmonary artery pressure, in contrast to short-term NO inhalation. BODY.ABBREVIATIONS: ANP = atrial natriuretic peptide; ARDS = acute respiratory distress syndrome; cGMP = guanosine 3',5'-cyclic monophosphate; CI = cardiac index; CO = cardiac output; DST = downslope time; EVLWI = extravascular lung water index; ICG = indocyanin green; ITBVI = intrathoracic blood volume index; MTT = mean transit time; NO = nitric oxide; PBVI = pulmonary blood volume index; RVEDVI = right ventricular end-diastolic volume index.

TITLE: Effectiveness of resistance training in combination with botulinum toxin-A on hand and arm use in children with cerebral palsy: a pre-post intervention study ABSTRACT.BACKGROUND: The aim of this pilot study was to examine the effects of additional resistance training after use of Botulinum Toxin-A (BoNT-A) on the upper limbs in children with cerebral palsy (CP). ABSTRACT.METHODS: Ten children with CP (9–17 years) with unilaterally affected upper limbs according to Manual Ability Classification System II were assigned to two intervention groups. One group received BoNT-A treatment (group B), the other BoNT-A plus eight weeks resistance training (group BT). Hand and arm use were evaluated by means of the Melbourne assessment of unilateral upper limb function (Melbourne) and Assisting Hand Assessment (AHA). Measures of muscle strength, muscle tone, and active range of motion were used to assess neuromuscular body function. Measurements were performed before and two and five months after intervention start. Change scores and differences between the groups in such scores were subjected to Mann–Whitney U and Wilcoxon Signed Rank tests, respectively. ABSTRACT.RESULTS: Both groups had very small improvements in AHA and Melbourne two months after BoNT-A injections, without differences between groups. There were significant, or close to significant, short-term treatment effects in favour of group BT for muscle strength in injected muscles (elbow flexion strength, p = .08) and non-injected muscles (elbow extension and supination strength, both p = .05), without concomitant increases in muscle tone. Active supination range improved in both groups, but more so in group BT (p = .09). There were no differences between the groups five months after intervention start. ABSTRACT.CONCLUSIONS: Resistance training strengthens non-injected muscles temporarily and may reduce short-term strength loss that results from BoNT-A injections without increasing muscle tone. Moreover, additional resistance training may increase active range of motion to a greater extent than BoNT-A alone. None of the improvements in neuromuscular impairments further augmented use of the hand and arm. Larger clinical trials are needed to establish whether resistance training can counteract strength loss caused by BoNT-A, whether the combination of BoNT-A and resistance training is superior to BoNT-A or resistance training alone in improving active range of motion, and whether increased task-related training is a more effective approach to improve hand and arm use in children with CP. BODY.BACKGROUND: Cerebral palsy (CP) is the most common cause of physical disability in childhood, with a prevalence of about 2.1 per 1000 live births [1]. Among these children, between 50–70% have impaired upper limb function [2-4]. According to the International Classification of Functioning, Disability and Health (ICF) framework [5], the causes and consequences of impaired upper limb function can be assessed at the levels of body function, activity, and participation. Spasticity, muscle weakness and impaired motor control are primary impairments at the level of body function for children with hemiplegic or diplegic CP. The primary impairments may give rise to secondary musculoskeletal complications such as contractures and deformities, again resulting in limited range of motion [6]. Activity limitations are presumed to result from these motor impairments and also from additional disturbances of sensation, perception, cognition, communication, and behaviour [7]. Spasticity, reduced muscle strength, and limited active supination range have been found to be related to hand and arm use [8], which may lead to difficulties performing several activities of daily living involving reaching, grasping, and manipulating objects. Consequently, much of the treatment offered is targeted towards treating neuromuscular impairments at the level of body functions, while intending to improve performance at the level of activity [9]. However, there is a lack of evidence as to what extent these treatments alter the motor prognosis or make a clinically significant change in hand and arm use at the level of activity in ICF [10,11]. Injections of Botulinum Toxin-A (BoNT-A) are often applied to improve impairments at the level of body functions, such as reducing spasticity, facilitating movement, and preventing secondary contractures, and ultimately improve hand and arm use in children with spastic CP [9]. The beneficial effect of BoNT-A injections in spastic lower limbs on gait has been well documented [12], but there is insufficient evidence with respect to improvements in hand and arm use [12,13]. A recent systematic review concluded that there is moderate evidence that BoNT-A injections alone are not effective in this respect and need to be combined with therapy to obtain functional gains [14]. However, the most effective treatment combinations remain unclear. BoNT-A in combination with resistance training is one treatment combination that warrants further investigation [14]. A few studies have investigated the combined effect of BoNT-A and intensive therapy with strength training as part of the intensive therapy [e.g., [15-17]]. However, these did not describe the procedures or measure the effect of strength training explicitly, making it difficult to evaluate whether such training has the potential to enhance the outcomes of BoNT-A injections. Previously it has been argued that only antagonists to spastic muscles should be strengthened in the BoNT-A effect period, during which spastic agonists have reduced muscle tone, in order to improve muscle balance across joints [9,18]. However, spastic muscles in the upper limbs have been found to show reduced strength during voluntary activation as well [19,20]. In addition, excessive weakness in injected spastic muscles is the most common adverse effect following BoNT-A treatment in the upper limbs [14]. Consequently, both the spastic muscles and their antagonists should be strengthened. The objective of this pilot study was to explore the effects of additional resistance training of spastic muscles and their antagonists on hand and arm use and neuromuscular body functions in children with CP after the use of BoNT-A. Hand and arm use were evaluated both according to capacity (what the child can do) with Melbourne assessment of unilateral upper limb function (Melbourne) [21,22], and according to performance (what the child spontaneously does) with Assisting Hand Assessment (AHA) [22,23]. Neuromuscular body functions were evaluated by testing active range of motion, muscle strength and muscle tone in spastic muscles and their antagonists in children with CP. BODY.METHODS: BODY.PARTICIPANTS: Participants were recruited from the outpatient records of the neuro-orthopaedic team at St. Olav’s Hospital (Trondheim, Norway). Children with CP were eligible to participate if they had: a) larger deficits in movement of one upper extremity in comparison to the other, b) functional use of the hands corresponding to Manual Ability Classification System (MACS) levels I or II [24], and an active grasp function in the involved extremity corresponding to category 5 (fair active assist) on the Modified House Functional Classification System [25], c) functional limitations when using the involved arm because of increased muscle tone in the forearm and elbow flexor muscles and a difference in active and passive range of motion, and d) were able to follow instructions and motivated to complete an eight week intensive resistance training program. Exclusion criteria were: a) treatment with BoNT-A in the upper extremities during the last six months, b) surgery on the upper extremity in the last two years prior to participation, and c) on-going intensive training. Thirty-one children were assessed and eighteen were found to be eligible to participate in the study. Eight of the eighteen refused to participate because they did not want BoNT-A injections. The remaining ten participants comprised nine children with hemiplegic and one with diplegic CP, all with MACS level II. These were matched in pairs based on age and assigned through tossing a coin to either a group receiving localized BoNT-A injections (group B, n = 5) or to a group receiving localized BoNT-A injections and resistance training (group BT, n = 5). Table 1 shows the demographic data for each of the groups. Table 1Demographic data for the groups  Group B (n = 5)Group BT (n = 5)Mean age (SD) 12 years (3.3 years) 14.8 years (3.0 years) Male/female (n) 4/1 1/4 CP type, hemiplegic/diplegic(n) 4/1 5/0 Group B = Botulinum Toxin-A injections. Group BT = Botulinum Toxin-A injections + eight weeks of resistance training. n = number of participants. SD = Standard deviation. The study was approved by the Regional Committee for Medical and Health Research Ethics. Written informed consent was obtained from the participants and parents before participation. Data were collected from September 2006 until March 2008. Participants were assessed on three occasions: before intervention (Baseline) and at two months (Post 2 months) and five months (Post 5 months) after the BoNT-A treatment, whether or not participating in resistance training. Baseline data from the study have been reported in a previous publication [8]. BODY.INTERVENTIONS: All the participants were instructed to continue their usual daily activities during the study period. None of the participants in group B performed specific training related to hand function, whereas those in group BT performed single-joint resistance training without additional functional training. All participants in groups B and BT received BoNT-A injections in m. pronator teres. In addition, three participants in group B and two in group BT received BoNT-A injections in m. biceps brachii and m. brachialis. Details of BoNT-A treatment are listed in Table 2. No participants received injections in spastic muscles in the hand or fingers because we wanted to avoid potential loss of grip strength [26] that might influence the possibility to perform the resistance training. BOTOX® from Allergan (Irvine, CA) was used (dilution 100 U/1 mL) and dosage was 25 or 50 U per injection sites, one site in pronator teres and brachialis, and two sites in biceps brachii. An experienced physician who was blinded for group assignment decided the dosage and performed the injections under general anaesthesia. A muscle stimulator was used in order to optimize accuracy of the injections of pronator teres [27]. Table 2Details of the Botulinum Toxin-A (BoNT-A) treatment for the participants in groups B and BT ParticipantGroup BGroup BTMuscle groups injected:Muscle groups injected:1 Pronator teres Pronator teres 2 Pronator teres, biceps brachii, brachialis Pronator teres 3 Pronator teres, biceps brachii, brachialis Pronator teres, biceps brachii, brachialis 4 Pronator teres, biceps brachii, brachialis Pronator teres, biceps brachii, brachialis 5 Pronator teres Pronator teres Pronator teres 25–50 units BOTOX® (Allergan, Inc.). Biceps brachii 50–75 units BOTOX® (Allergan, Inc.). Brachialis 25–50 units BOTOX® (Allergan, Inc.). The participants in group BT trained three times a week for eight weeks under supervision of a trained physiotherapist not involved in the assessments. Participants exercised alone or in pairs. The resistance training program followed guidelines from the National Strength and Conditioning Association [28] and consisted of ten minutes warm up and 30–40 minutes core strengthening and single-joint resistance training (using hand-held, free weights) for strengthening of elbow flexors and extensors, forearm pronators and supinators, and wrist flexors and extensors. Grip force was trained as well using exercise balls with increasing resistance. The intensity was set on the basis of 10 repetition maximum strength tested at baseline [29]. Once the participants were able to perform three sets of ten repetitions in an exercise, intensity was built up progressively on an individualized basis by increasing the weights by 0.25 – 0.5 kg. There was a one-minute recovery period between all sets and a two-minute recovery period between different exercises. BODY.OUTCOME MEASURES: Hand and arm use were evaluated with Melbourne [21] and with AHA [23]. Melbourne measures unimanual capacity in tasks that simulate everyday activities. The children are asked to perform their best in 16 tasks that are scored according to 37 sub-scores. Percentage scores (0–100) were calculated and used for analyses [21]. Melbourne has been found to be a reliable and valid instrument for measuring quality of upper limb function, and the smallest detectable difference to measure real change needs to be at least 12% [30]. A randomized selection of 3–15 items from seven different subjects was scored by a second therapist. The intra-class correlation coefficient for the consistency of scorings between therapists was high (ICC = 0.96). AHA is a Rasch-built instrument that measures how children spontaneously use their involved upper limb in bimanual tasks (i.e. bimanual performance) without instructing the child to perform at their best [23]. The AHA was scored on 22 items with a four-point rating scale (22–88 raw sore). Scores on AHA were converted into equal interval logits (log odds probability units), which were converted to a 0–100 AHA-unit scale [31]. The smallest detectable difference for AHA is 5 AHA-units [32]. AHA has been found to be valid and reliable [33] and has been validated for children up to 12 years. In our study the participants were up to 17 years. However, no association between age and AHA-units was found in the current study (rs =.202, p=.576), nor in a previous study by our research group [8] with similar age range. The Baseline and Post 2 months AHA-unit scores for the participants were compared to blinded scorings made by an occupational therapist not involved in the treatment or data collection. The intra-class correlation between the two sets of scorings was high (ICC = 0.89). Neuromuscular body functions were evaluated by testing active range of motion, muscle strength and muscle tone in spastic muscles. Active range of motion of the elbow and forearm was measured using a mechanical goniometer following standard procedures [34]. A stationary dynamometer BIODEX System 3 Pro (Biodex Medical Systems, Shirley, NY, USA), found to be reliable in the lower extremities of children with CP [35,36], was used to evaluate muscle tone and strength in the elbow and forearm. Measurements in the elbow were performed with the forearm in neutral position and those in the forearm with the elbow in 90° flexion. The shoulder was slightly abducted in all measurements. Muscle tone was evaluated as resistance to passive movement during three trials at two velocities, 10°/s and 180°/s, in elbow extension and forearm supination. Strength was evaluated during three dynamic maximal voluntary contractions (MVC) at a velocity of 60°/s. One practice trial was performed first to ensure that the task was performed correctly. There was a one-minute break between the trials. The procedure was first carried out on the forearm, then on the elbow. Data analysis was carried out using Matlab (The Mathworks Inc., Natick, MA, USA), version 7.6. Prior to further analyses, torque signals were low-pass filtered with a cut-off frequency of 6 Hz. The torque recorded during the passive movements at 10°/s was used to correct the other torque measures for arm weight. The peak resistance torque during 180°/s in the trial with the least resistance was used to reflect muscle tone in the elbow flexors and forearm pronators [35]. The highest peak torque value during the three isokinetic contractions was used as strength parameter. Isometric grip force was measured using Grippit® (AB Detektor, Göteborg) in the standardized position recommended by the American Society of Hand Therapists for hand-grip dynamometry [37]. Grippit has been found to be a reliable instrument for measuring peak grip strength in children of different age groups [38]. Three maximal trials were performed for each hand, with a two-minute rest period between repetitions. The highest peak value during the trials was used for further analyses. BODY.STATISTICAL ANALYSIS: Data were analysed using SPSS, version 18.0 (SPSS Inc., Chicago, IL, USA). Non-parametrical analyses were applied because of the small sample size and deviation from a normal distribution for several of the variables. Within-group differences were tested using Wilcoxon Signed Rank Test, and between-group differences by means of Mann–Whitney U test for Baseline and in change scores between Baseline to Post 2 and Post 5 months, respectively. Post 2 months and Post 5 months were tested separately against Baseline in order to reduce the effect of missing data. Significance level was set at p < .05 and trends at p < .1 are reported. Post-hoc power calculation suggested that given the sample size, our study had 80% power to detect a difference in AHA of three units from Baseline to Post-intervention with a p-value < .05. BODY.RESULTS: All participants completed the intervention period and the subsequent Post 2 months assessment. One participant in group BT dropped out of the Post 5 months assessment. At Baseline, the only significant group difference was higher forearm supination resistance torque in group B than in group BT (p = .05). Despite differences in gender distribution between the groups, there were no differences for any of the strength parameters at baseline. Results for the hand and arm use measures at Baseline, Post 2 and Post 5 months are shown in Figure 1. Both groups had small improvements in hand and arm use. However, none of the changes were statistically or clinically significant. For group B, the median change score from Baseline to Post 2 and Post 5 months was 1 AHA-unit, while the results for group BT were 1 and 0.5 AHA-unit respectively. The equivalent results from Melbourne for group B were 1.7 and 0.8% change, and for group BT 0.8 and 0.4% change. There were no between-group differences. Figure 1Hand and arm use. Box-plots for hand and arm use measured with the Assisting Hand Assessment (AHA) (a) and the Melbourne Assessment for Unilateral Limb Function (b). Baseline Post 2 months Post 5 months measures’ with Baseline (solid/dark grey) Post 2 months (striped grey) Post 5 months (dotted/light grey) measures for group B (receiving Botulinum Toxin-A) and group BT (receiving Botulinum Toxin-A + 8 weeks of resistance training). Boxes are inter-quartile ranges, the solid horizontal line is the median, whisker bars are the extreme values. Outliers are shown as circles. Findings for the strength measures at Baseline, Post 2 and Post 5 months are shown in Figure 2. All participants in group B lost pronation strength (p = .043), and the majority also experienced small, non-significant strength losses for all other strength parameters (elbow flexors: n = 4, elbow extensors: n = 3, supinators: n = 4, and grip strength: n = 3). In group BT, all participants increased their grip strength (p = .043). Otherwise there were no significant changes, although most children (n = 4) gained strength in elbow flexors, elbow extensors, and supinators, whereas pronation strength decreased in 3 children. When comparing the groups, there were significant or close to significant differences in treatment effects from Baseline to Post 2 months for all strength parameters (p’s = .016–.076) except for pronation strength. From Baseline to Post 5 months, the only significant difference in treatment effect between the groups was in grip strength (p = .050). Otherwise, both groups were more or less back at their Baseline values. Figure 2Strength measures. Box-plots for elbow flexion (a) and extension (b), and forearm supination (c) and pronation (d) voluntary peak torque in Newtonmeter (Nm), and grip force (e) in Newton (N). Baseline Post 2 months Post 5 months measures’ with Baseline (solid/dark grey) Post 2 months (striped grey) Post 5 months (dotted/light grey) measures for group B (receiving Botulinum Toxin-A) and group BT (receiving Botulinum Toxin-A + 8 weeks of resistance training). Boxes are inter-quartile ranges, the solid horizontal line is the median, whisker bars are the extreme values. Outliers are shown as circles. Significant group differences in treatment effect are indicated. The muscle tone results, measured as resistance to passive elbow extension and forearm supination, are shown in Figure 3. In both groups there was only a small, non-significant decrease or no change in elbow flexor muscle tone from Baseline to Post 2 and Post 5 months, with no between-group differences in treatment effect. Forearm supination resistance decreased in both groups, but considerably more so in group B (p = .043), resulting in a significant difference in treatment effect between the groups (p = .047). Figure 3Muscle tone measures. Box-plots for elbow extension resistance torque and forearm supination resistance torque in Newtonmeter (Nm). Baseline Post 2 months Post 5 months measures’ with Baseline (solid/dark grey) Post 2 months (striped grey) Post 5 months (dotted/light grey) measures for group B (receiving Botulinum Toxin-A) and group BT (receiving Botulinum Toxin-A + 8 weeks of resistance training). Boxes are inter-quartile ranges, the solid horizontal line is the median, whisker bars are the extreme values. Outliers are shown as circles. Significant group differences in treatment effect are indicated. The results from active forearm supination range at Baseline, Post 2 and Post 5 months are shown in Figure 4. All participants in group BT improved their supination range with a median change of 27.5° (p = .043 from Baseline to Post 2) and 10° (p = .066 from Baseline to Post 5). For group B, the results were less consistent. At both post-tests, two children showed improved range, two had not changed, and one showed decreased range, giving a median change in active supination range of 0°. Comparing the two groups, there was a statistical trend in favour of group BT (p  Figure 4Forearm supination. Box-plot for active forearm supination range in degrees (deg.). Baseline Post 2 months Post 5 months measures’ with Baseline (solid/dark grey) Post 2 months (striped grey) Post 5 months (dotted/light grey) measures for group B (receiving Botulinum Toxin-A) and group BT (receiving Botulinum Toxin-A + 8 weeks of resistance training). Boxes are inter-quartile ranges, the solid horizontal line is the median, whisker bars are the extreme values. Outliers are shown as circles. Significant group differences in treatment effect are indicated. BODY.DISCUSSION: This pilot study explored the effects of resistance training in addition to BoNT-A treatment on hand and arm use and neuromuscular body functions in a small sample of children with CP. Our results indicate that the addition of eight weeks resistance training strengthens non-injected muscles temporarily, and may possibly reduce the short-term strength loss that results from BoNT-A injections in the spastic muscles, without a concomitant systematic increase in muscle tone. Furthermore, additional resistance training may increase active range of motion to a larger extent than BoNT-A alone. However, none of the improvements in neuromuscular body functions further improved hand and arm use at the level of activity in ICF. After eight weeks of intensive training, median strength improvements of 32 to 50% were observed in non-injected antagonists to the spastic muscles (range elbow extensors: −14 to 42% and range supinators: −58 to 72%). This is similar to strength improvements reported previously in the upper extremities [17,39]. However, our results demonstrate large variations in the effect of resistance training in this small sample, and some of the participants experienced strength losses despite resistance training. It can therefore be speculated that some children with CP might benefit more from resistance training than others. Based on a review of randomised controlled trials in the lower limbs, it has recently been suggested that not all children with CP respond to resistance training to the same extent [40]. Age and severity might have an effect, and the degree of selective motor control might also affect the response to training for some children with unilateral CP [40]. Larger studies are needed to further investigate this suggestion, using analytical approaches to determine the sources of variability. BoNT-A injections alone resulted in a significant decrease in muscle tone, but also in a temporary strength loss in injected spastic muscles, similar to findings reported by others [14,17,26]. Despite decreased muscle tone, the ability to generate force in the antagonists to the injected spastic muscles did not show even a trend towards improvement in the participants receiving BoNT-A treatment only. This indicates that spastic activity does not limit strength in the antagonist to the spastic muscle, at least not at an isokinetic velocity of 60 °/sec. Additional resistance training improved muscle strength in injected agonists in three children, and it is therefore possible that strength losses associated with BoNT-A injections may be counteracted by progressive resistance training following approved guidelines. Larger clinical trials are needed to further illuminate these results, especially since several of the children in our study were still weakened in the injected muscles despite resistance training. Consequences of weakening injected muscles should therefore be considered when using BoNT-A [17]. In line with current literature [40,41], no systematic increase in muscle tone was found following resistance training. Active supination range improved more in the group who performed resistance training in the BoNT-A effect period, and seems to be more effective than BoNT-A alone, showing a trend approaching significance. Strengthening of antagonists to spastic muscles in combination with reduction of muscle tone in spastic agonists is a recommended approach [9], but few studies have addressed the combined effect thoroughly [14]. Most studies have evaluated differences in passive range of motion following BoNT-A injections and therapy and found no effect [14]. One recent study found similar improvements in active elbow extension and wrist flexion between therapy with and without BoNT-A [17], whereas another study concerned the effect of BoNT-A in combination with therapy compared to therapy alone, and found larger improvements in active supination for therapy alone [16]. However, the latter finding could be attributed to differences between the groups at baseline. Active supination range has been found to be a significant predictor for activity in the upper extremities [8], and finding optimal interventions is thus important. It should therefore be investigated whether or not resistance training alone is more effective in improving active supination range than resistance training in combination with BoNT-A, since our results indicate that muscle weakness constrains active movement to a larger extent than spasticity. Grip strength and muscle strength in antagonists to the spastic muscles (elbow extensors and forearm supinators) improved significantly more in the group who performed resistance training in combination with BoNT-A, but did not further improve unimanual or bimanual hand and arm use compared to BoNT-A alone. Similar results were obtained in a recent study by Rameckers and co-workers [17] showing that increased muscle strength and accuracy do not necessarily transfer to improved use of the affected hand in fine motor activities as measured with Melbourne. There might be several reasons for this lack of transfer. To begin with, our sample size is small and possibly a larger sample would have revealed other findings. However, none of the participants in our study had improvements of 5 AHA-units or more that is reported to be the smallest detectable difference to measure real change [32]. A second possibility is that strength gains in our study were not large enough to engender a meaningful change in hand and arm use, or that strength gains transfer to other functions than those measured with Melbourne and AHA. AHA is thought to reflect how a child performs in his or her usual environment [42], but no test performed in a standardized environment can fully capture how a child performs activities in daily life. Future studies should therefore include this aspect. A final explanation for the lack of carry-over to functional tasks could be specificity of training. In particular, for children who are unilaterally affected in the upper limbs, it may be due to problems with motor planning [43] and potential non-use of the affected hand [44], which could offset gains at the level of body function. This is in accordance with Damiano [45] who proposed that treating the impairment alone may be too far removed from the functional tasks we want to improve. The use of task-related resistance training following single-joint resistance training may have greater impact on the use of the affected hand in bimanual activities [10] and would possibly also have more lasting effects on muscle strength. In our study, none of the strength improvements were long-lasting. If the participants had integrated and transferred the improved muscle strength to improved hand and arm use, there would be a greater possibility for longer lasting effects also on muscle strength. Moreover, it is more likely that the improved limb functions will be implemented in activities when these activities are explicitly described as individual goals at the start of therapy. In addition to the small sample size, the present study has some other limitations. Assignment to the different interventions was done by matching the children in pairs followed by tossing a coin, a method that has been found to be sensitive to selection bias. Nonetheless, the procedure resulted in two groups with similar baseline assessments, except for pronation resistance torque and gender. The difference in gender between the groups could have confounded the results if treatment effect would depend on gender. So far, however, no indications of such a gender effect are present in the literature, but caution is warranted. Moreover, the National Strength and Condition Association [28] states in their updated position statement paper on youth resistance training that there is no clear evidence of any major difference in strength between boys and girls in preadolescents. Thus, we consider it unlikely that gender is a confounder in this study. Although difficulty performing daily activities was not included as a criterion for inclusion, AHA and Melbourne indicated that all participants had limitations in the use of the involved hand. BODY.CONCLUSIONS: It has previously been found that muscle strength and active supination range are related to hand and arm use. It is therefore important to investigate whether treatments targeting these impairments at the level of body function will have an effect on the level of activity. We found that resistance training in combination with BoNT-A did not improve hand and arm use further compared to BoNT-A alone, even though grip strength and strength in antagonists to spastic muscles improved significantly more for the BT group. Specificity of training and strength gains could be the explanation for this lack of transfer. In future studies, there is a need for more insights into whether the use of more task-related resistance training, alone or in combination with single-joint resistance training, has greater impact on the use of the affected hand in bimanual activities, and results in longer-lasting strength effects than only the general resistance training employed in the current study. Resistance training improved strength temporarily in non-injected muscles and may possibly compensate for the temporary strength loss in injected spastic muscles due to BoNT-A treatment, without a concomitant increase in muscle tone. However, larger clinical trials are needed to further investigate whether strength losses associated with BoNT-A may be counteracted by resistance training. Furthermore, it should be investigated whether resistance training in addition to BoNT-A injections improves active range of motion to a larger degree than resistance training alone. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: AKGE: substantial contributions to the conception and design, acquisition of data, analysis and interpretation of data, drafting and revising the article critically. SMB: substantial contributions to the conception and design, acquisition of data, analysis and interpretation of data, drafting and revising the article critically. RS: substantial contributions to the conception and design, acquisition of data, and revising the article critically. TL: substantial contributions to the conception and design, and revising the article critically. BV: substantial contributions to the conception and design, data analysis and interpretation of data, drafting and revising the article critically. KR: substantial contributions to the conception and design, acquisition of data, analysis and interpretation of data, drafting and revising the article critically. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2431/12/91/prepub

TITLE: Daily intake of rosehip extract decreases abdominal visceral fat in preobese subjects: a randomized, double-blind, placebo-controlled clinical trial ABSTRACT.BACKGROUND: Obesity has become a great problem all over the world. We repeatedly screened to find an effective food to treat obesity and discovered that rosehip extract shows potent anti-obesity effects. Investigations in mice have demonstrated that rosehip extract inhibits body weight gain and decreases visceral fat. Thus, the present study examined the effect of rosehip extract on human body fat in preobese subjects. ABSTRACT.METHODS: We conducted a 12-week, single-center, double-blind, randomized, placebo-controlled study of 32 subjects who had a body mass index of ≥25 but <30. The subjects were assigned to two random groups, and they received one tablet of placebo or rosehip that contained 100 mg of rosehip extract once each day for 12 weeks with no dietary intervention. Abdominal fat area and body fat percent were measured as primary outcomes. The other outcomes were body weight and body mass index. ABSTRACT.RESULTS: Abdominal total fat area, abdominal visceral fat area, body weight, and body mass index decreased significantly in the rosehip group at week 12 compared with their baseline levels (P<0.01) after receiving the rosehip tablet intake, and the decreases in these parameters were significantly higher when compared with those in the placebo group. Additionally, body fat percent tended to decrease compared with the placebo group and their baseline level. Moreover, the abdominal subcutaneous fat area was significantly lower in the rosehip group than in the placebo group at week 12 after the initiation of intake (P<0.05). In addition, there were no abnormalities, subjective symptoms, and findings that may indicate clinical problems during the study period. ABSTRACT.CONCLUSION: These results suggest that rosehip extract may be a good candidate food material for preventing obesity. BODY.INTRODUCTION: Obesity is one of the most important diseases and has become a global health problem. It is caused when a balance between energy intake and expenditure is lost and is characterized as a state of increased body weight by excessive lipid accumulation in the white adipose tissue.1 Hypertrophied fat cells can increase a risk of type 2 diabetes associated with insulin resistance. Obesity can cause dysbolisms such as hyperlipidemia, hypertension, and arteriosclerosis, and these disorders can induce fatal cardiovascular diseases.2 Therefore, the prevention and improvement of obesity, particularly decrease of visceral fat is important in the control of these metabolic diseases. Various functional foods or natural components have been recently developed for preventing or improving obesity as the alternative means of lifestyle improvement or medical treatment for obesity. As the result of our screening, we found that tiliroside, a major rosehip seed constituent, has fat metabolism acceleration and glucose clearance improvement effects.3 Rosehip, the whole fruit of Rosa canina L. belonging to the family Rosaceae, is popular all over the world. In Europe and America, rosehip is processed widely into jam and juice, used as a vitamin C supplement, in cosmetics, and as a folklore drug in the form of tisane.4,5 The functional capacities of rosehip includes efficacy on diuresis, as a laxative, and as treatments for gout and rheumatism.6,7 There have also been reports of several effects including antioxidative,8 anticold, and anti-inflammatory actions,9 inhibitory effects on metabolism of arachidonic acid and formation of cyclooxygenase 1, cyclooxygenase 2, and leukotriene B4,10 suppression of inflammation and cancer cell proliferation attributable to anticomplementary effect,11 antibacterial action and suppression of experimental nephrolithiasis,12 melanin production inhibition,13 and antidiarrheal action using a methanol extract of rosehip leaves.14 Large-scale clinical studies have confirmed the efficacy of rosehip in treating human knee joint arthropathy.15 It is well known that rosehip contains an abundance of vitamin C and polyphenols.16 Especially, tiliroside, a major glycosidic flavonoid isolated from rosehip seed, has a variety of pharmacological activities, eg, an anticomplementary effect,17 a hepatic protection action,18 an anti-inflammatory and antioxidative action,19 an inhibitory effect on the depression of expression of insulin-like growth factor-1 and hypoxia-inducible factor-1 due to oxidative stress,20 and an antibacterial action.21 Recently, the antiobesity effects of rosehip have been clarified by in vitro and in vivo studies. Ninomiya et al showed that an 80% aqueous acetone extract of the whole fruit of R. canina L. significantly suppressed body weight gain and prevented increases in the visceral fat in nonobese mice without any changes in diet intake.3 They also reported that tiliroside upregulated the expression of peroxisome proliferator-activated receptor α messenger RNA in the liver. We showed that an aqueous ethanol extract of rosehip inhibited lipid accumulation in adipocytes using 3T3-L1 cells and diet-induced obese mice.22 Andersson et al reported that rosehip juice decreased the systolic blood pressure and plasma cholesterol levels in obese subjects.23 However, no clinical trials have evaluated the antiobesity effects of rosehip. Obesity is defined by the World Health Organization classification as a body mass index (BMI) ≥30 kg/m2, where a person with a BMI ≥25 kg/m2 but <30 kg/m2 is classified as preobese.24 In addition, based on the diagnostic criteria for obesity provided by the Japan Society for the Study of Obesity in 2011, an abdominal visceral fat area $100 cm2 is an important factor in obesity-related metabolic disorders.25 In the present study, we targeted preobese subjects and confirmed the antiobesity effects of rosehip extract, where a reduction in the abdominal body fat area and body fat percent were the primary outcome. BODY.MATERIALS AND METHODS: BODY.STUDY DESIGN: A single-center, double-blind, randomized, placebo-controlled study was designed to evaluate the efficacy of rosehip extract. All experiments conformed to the Helsinki Declaration (adopted in 1964 and amended in 2008) and were conducted under the control of the principal investigator. The study protocol and all related documents were approved by Miyawaki Orthopedics Clinic Institutional Review Board. Appropriate explanations were provided to the subjects for important factors including the study purpose, contents, methods, and predicted adverse reactions. Informed consent was voluntarily obtained from each subject in writing. The study was performed in Fukuhara Clinic in Eniwa, Hokkaido, Japan, from August 2011 to December 2011. BODY.SUBJECTS: In total, 152 subjects agreed to participate in the study, who were paid volunteers enrolled by the New Drug Research Center Inc. (Previously known as: New Drug Development Research Center Inc.), and 32 subjects (16 males and 16 females) who met the following inclusion and exclusion criteria were selected by screening (Figure 1, Table 1). The 32 subjects were assigned to two equal groups to avoid bias related to sex, BMI, and body fat percentage. BODY.MATERIALS: Rosehip extract (Rosehip Polyphenol EX™, Morishita Jintan Co, Ltd, Osaka, Japan) comprised an aqueous ethanol extract of rosehip containing its seeds, dextrin, and cyclodextrin. This extract contains not less than 0.1% of tiliroside. The rosehip tablet contained 100 mg of rosehip extract and excipients, and the placebo tablet was indistinguishable from the rosehip tablet. The compositions and nutritional contents of both the tablets are shown in Table 2. BODY.STUDY SCHEDULE AND MEASUREMENTS: All subjects received one rosehip or placebo tablet once a day for 12 weeks by chewing, and they visited the clinic for assessments and measurements at the start of intake (W0) and on the 4th (W4), 8th (W8), and 12th week (W12). The subjects were allowed to drink only water after 9 pm on the day before the visit for examinations. During the study period, the subjects were not permitted to use medical products or foods with lipid-lowering or body fat-reducing actions, and we instructed the subjects not to change their life patterns greatly compared with that before the initiation of the study. All subjects were assessed to determine their physical parameters (body weight, body fat percentage, BMI, body temperature, blood pressure, and heart rate), hematological parameters (white blood cells, red blood cells, hemoglobin, hematocrit, and platelets), blood biochemical parameters (total protein [TP], albumin, albumin/globulin ratio, aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transpeptidase, fasting blood sugar, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglyceride, uric acid, urea nitrogen, creatinine, Na, K, Cl, and Ca), urinalysis (urine protein, urine glucose, urobilinogen, bilirubin, ketone body, occult blood, specific gravity, and pH), and abdominal visceral and subcutaneous fat areas. All biochemical analyses were performed in Daiichi Kishimoto Clinical Laboratory Co, Ltd (Hokkaido, Japan) using the automatic analyzers XE-2100 (Sysmex Corp, Hyogo, Japan) for hematological analysis, JCA-BM8060 (JEOL Ltd, Tokyo, Japan) for blood biochemical analysis, and AX-4280 (Arkray, Inc., Kyoto, Japan) for urinalysis. BODY.DIETARY RECORD AND PHYSICAL ACTIVITY: To determine the energy intake and consumption, we collected meal survey slips where the subjects described their food content and the number of steps counted by a pedometer for 3 days prior to each visit at W0, W8, and W12, and we analyzed the data according to the procedure described by Maki et al.26 Based on the meal records, a nutritionist calculated the energy intake and the amount of carbohydrates, fat, and proteins consumed. The energy consumption was calculated as the sum of the basal metabolic rate and kinetic energy. The basal metabolism is usually calculated as the “basal metabolism standard value × body weight”, but in this study, because the basal metabolism becomes excessive in obese subjects, it was calculated using the ideal body weight based on the height. Further, based on the assumption that the kinetic energy was 3.3 metabolic equivalents for males and 3.0 metabolic equivalents for females, the energy consumption (kcal) was calculated by multiplying the walking time from the number of steps by the kinetic energy (metabolic equivalents), body weight, and 1.05 (kcal/kg/h).27 BODY.PHYSICAL ASSESSMENT: Body weight was measured using the normal method, and BMI was calculated as body weight/height2. Body fat percentages were determined using body composition analyzer (TBF-310, Tanita Corp, Tokyo, Japan). Computed tomography (CT) scans were performed using a CT-W450 CT scanner system (Hitachi Medical, Tokyo, Japan). Abdominal body fat areas (subcutaneous and visceral fat areas) were calculated based on an abdominal CT scan image using visceral fat measurement software (Fat Scan™ Ver. 3.0, N2 Systems Inc., Osaka, Japan). The total fat area was calculated as the sum of the subcutaneous and visceral fat areas. To consider the risk of radiation exposure in subjects, CT scans were performed only on W0, W8, and W12. BODY.SAFETY ASSESSMENT: The subjects entered their subjective symptoms in a subject diary. At W0, W4, W8, and W12, the investigator conducted a medical interview with the subjects based on the subject diary and the health status of the subjects was examined by auscultation and percussion. Body temperature, blood pressure, and pulse were measured at W0, W4, W8, and W12, and hematology tests, blood biochemical examinations, and urinalysis were performed at W0, W8, and W12. BODY.STATISTICAL ANALYSIS: Mean values and standard deviations of each datum were calculated for each group. A paired t-test was used to compare the data before and after the intake within a group. The confirmation of homoscedasticity between the placebo and the rosehip groups was conducted using the F-test. If the homoscedasticity was verified, a Student’s t-test was used, whereas Welch’s t-test was used for comparisons if the variances were unequal. In addition, a signed rank sum test was used for intergroup comparisons of the semiquantitative urine values before and after the intake, and Wilcoxon’s rank sum test was used for comparisons between the placebo and test food groups. A P value less than 5% was considered to be significant. Statistical analysis was performed using Microsoft Excel® 2003 (Microsoft Corp, Redmond, WA, USA) and SAS® 9.1.3 Foundation (SAS Institute, Cary, NC, USA). BODY.RESULTS: The test food intake rates for the placebo group and the rose-hip group at 12 weeks were 99.5% and 99.6%, respectively. No subject left during the study period, and no outliers were observed for any parameter. Thus, all data were subjected to statistical analysis. Table 3 shows the background data for the subjects. The abdominal total fat and abdominal visceral fat areas were somewhat higher in the control food group compared with the test food group; however, no significant difference was observed between the two groups (P=0.32 for the abdominal total fat area and P=0.21 in the abdominal visceral fat area). In addition, there were no significant differences between the two groups in terms of the other parameters. Table 4 represents the energy intake and consumption, which was calculated based on the number of steps counted and the basal metabolism. No significant intragroup and intergroup differences were observed; thus, there were unlikely to have been any major changes in the lifestyles of the subjects during the study period. The results of the physical assessment of the placebo group and the rosehip group are shown in Table 5. The measurement values at W12 were compared with those at W0, which showed significant decreases in the body weight and BMI in the rosehip group; the decreases in the body weight and BMI were significantly larger at W12 in the rosehip group than in the placebo group. Moreover, at W12, the body fat percentage was lower in the rosehip group compared with that in W0, but the difference was not significant. At W8 and W12, the abdominal visceral fat area in the rosehip group significantly decreased compared with the area at W0, and the decrease in the area at W12 was significantly larger than that in the placebo group. However, at W12, the abdominal total fat area in the rosehip group was significantly lower than that at W0, where the decrease was significantly larger than that in the placebo group. In contrast, there was no significant intragroup variation in the abdominal subcutaneous fat area, but the decrease in this area in the rosehip group at W12 was significantly larger than that in the placebo group. Further, blood pressure was measured using an automated sphygmomanometer, and pulse rates were measured at the same time as the blood pressure. The results of the measurement are also shown in Table 5. The blood biochemical parameters, hepatic functional markers, fasting blood sugar, and serum lipids, which are cardiovascular disease risk factors, and other parameters, were measured (Table 6). In the rosehip group, TP (W8), fasting blood sugar (W8), high-density lipoprotein cholesterol (W12), and systolic blood pressure (W4) changed significantly from W0, but the variations were within the normal physiological range. In the rosehip group, the following adverse events were recorded: common cold in four cases, headache in four cases, pyrexia and cough in two cases each, and sore throat, congested nose, loss of appetite, and abdominal pain in one case each. Furthermore, the adverse events observed in the placebo group were common cold in five cases, headache in three cases, pyrexia/cough in one case, and abdominal pain in one case. All these events were transient, and there was no observed aggravation due to the continuous intake of the placebo and the rosehip tablet in each group. There were no clinically problematic abnormalities or findings according to the physical assessments, blood biochemical and hematological parameters (Table 7), urinalysis (Table 8), and the medical interview by the investigator, which included auscultation and percussion. BODY.DISCUSSION: The World Health Organization published diagnostic criteria for a syndrome called “metabolic syndrome” in 1998, and there have been extensive subsequent discussions/reviews on these diagnostic criteria.28 Metabolic syndrome is a state that combines the risk factors of high blood sugar, high blood pressure, and hyperlipidemia due to visceral fat type obesity (visceral fat obesity/abdominal obesity). Study groups in various countries have reported that the cardiovascular disease risk is increased in subjects with metabolic syndrome.29–31 Frequent attempts have been made to lower the risk of cardiovascular diseases by decreasing visceral fat in studies of obese subjects. The “VACATION-J study” of Japanese subjects showed that an increase in visceral fat heightened the risk of cardiovascular diseases.32 In addition, a direct correlation was reported between decreased visceral fat and improvement in cardiovascular disease risk factors, including high blood pressure, hypo-high-density lipoprotein cholesterolemia, hyper-low-density lipoprotein cholesterolemia, hypertrig-lyceridemia, and fasting hyperglycemia.33 Czernichow et al reported that abdominal obesity rather than BMI is related to death risk due to cardiovascular diseases.34 Matsushita et al conducted a study of 6,292 Japanese subjects (5,606 males with a mean BMI of 24.1 kg/m2 and 686 females with a mean BMI of 23.0 kg/m2), which confirms that the abdominal visceral fat area is a better index of metabolic syndrome than the abdominal subcutaneous fat area and waist circumference.35 In an analysis of the effects of rosehip on cardiovascular disease risk factors, Andersson et al showed that a rosehip supplement suppressed the progression of diabetes in C57BL/6J mice and that downregulation of the lipogenic program in the liver was one of the mechanisms related to this effect.36 They also examined the effects of rosehip juice on cardiovascular disease risk markers for type 2 diabetes and circulatory diseases using a randomized, double-blind, crossover method with obese human subjects.23 Based on the reports described earlier, we planned and conducted a clinical study to determine the antiobesity effects of rosehip extract. Obtained results suggest that rosehip extract could lead to weight loss, reducing the risk of cardiovascular disease. The rosehip extract used in this study is commercially available as a functional food ingredient named “Rosehip Polyphenol EX”, which includes ≥0.1% tiliroside as an active component. Prior to starting the study, we confirmed that tiliroside content in the rosehip extract was 0.12%. We showed that an aqueous ethanol extract of rosehip inhibits lipid accumulation in the adipocytes.22 In addition, Ninomiya et al administered 0.1–10 mg/kg of tiliroside to mice for 2 weeks and observed the suppression of body weight gain, decreased amount of visceral fat, and they also found that peroxisome proliferator–activated receptor messenger RNA was upregulated in the liver of tiliroside-administered mice.3 Furthermore, Goto et al demonstrated that tiliroside stimulates fatty acid oxidation in obesity model KK-Ay mice.37 These reports indicate that the antiobesity effects of rosehip extract or tiliroside depend on the inhibition of lipid accumulation in adipose tissue and the stimulation of fatty acid oxidation. The rosehip tablet used in this study contained 0.1 mg of tiliroside in one tablet. Thus, we expect that tiliroside contributed to the reduction of visceral fat by the rosehip extract, and its effective dose seems very low. The autonomic nervous system (ANS) participates in the regulation of energy metabolism, and it plays an important role in body weight maintenance.38,39 ANS activity can be quantified based on the power spectral analysis of the heart rate variability.40,41 A preliminary study showed that the rosehip extract stimulates ANS activity and tends to increase the fat utilization at rest and during exercise based on power spectral analysis of the heart rate variability and an analysis of expired gas in 25 healthy male and female adults. In addition, the visceral fat is more reactive than the subcutaneous fat to lipolysis by catecholamine.42,43 In the present study, the abdominal visceral fat area was reduced earlier than the subcutaneous fat area. Hence, it is possible that the fat combustion effect induced by enhanced ANS activity is involved in the body fat-lowering effect of the rosehip extract. It is well known that the body weight and body fat mass are affected by the daily diet and exercise regimens. Thus, we monitored the energy intake and consumption of the subjects, but there were no major changes in the lifestyles of the subjects during the study period. Therefore, the changes detected in the present study were attributable to the rosehip extract, rather than lifestyle changes. The safety of rosehip extract has already been demonstrated by a single-dose toxicity test, a 30-day repeated-dose toxicity test, and an Ames test (unpublished data). In this study, the results of the safety outcomes, subjective symptoms, and medical assessments demonstrated that no serious adverse events were caused by the test food during the study period. Furthermore, rosehip extract does not interact with nifedipine, a cytochrome P450 3A substrate, according to a pharmacokinetics study in rats (unpublished data). However, a previous study reported that rosehip fruit juice delayed the peak time required to attain the maximum blood concentration (Tmax) and there was a 16% decrease in the area under the blood concentration–time curve with paracetamol.44 Thus, the rosehip extract is generally recognized as safe, but care should be taken when patients who are receiving medication also use rosehip. In conclusion, the results of the present study demonstrate that rosehip extract may be useful as a supplement to safely reduce abdominal visceral fat in preobese subjects. Therefore, it is anticipated that rosehip extract will reduce the risk of cardiovascular disease.

TITLE: Comparison of the trapezius and the adductor pollicis muscle as predictor of good intubating conditions: a randomized controlled trial ABSTRACT.BACKGROUND: Adequate muscle relaxation is important for ensuring optimal conditions for intubation. Although acceleromyography of the adductor pollicis muscle is commonly used to assess conditions for intubation, we hypothesized that acceleromyography of the trapezius is more indicative of optimal intubating conditions. The primary outcome was the difference between both measurement sites with regard to prediction of good or acceptable intubating conditions. ABSTRACT.METHODS: Neuromuscular blockade after injection of rocuronium 0.3 mg/kg IV was measured simultaneously with acceleromyography of the adductor pollicis muscle and the trapezius muscle in sixty female patients, American Society of Anesthesiologists physical status I to III, undergoing general anesthesia for gynecologic surgery. Exclusion criteria were: expected difficult tracheal intubation (e.g. history of difficult intubation, reduced mouth opening (< 2 cm) and/or Mallampati Score 4), increased risk of pulmonary aspiration (e.g. gastroesophageal reflux or delayed gastric emptying) allergies to drugs used during the study, pregnancy, neuromuscular diseases, medication with potential to influence neuromuscular function (e.g. furosemide, magnesium, cephalosporins) and hepatic or renal insufficiency (serum bilirubin >26 μmol/L, serum creatinine >90 μmol/l). Patients were randomized to 2 groups: group A (n = 30): endotracheal intubation after onset of the neuromuscular block at the adductor pollicis muscle. Group B (n = 30): endotracheal intubation after onset at the trapezius muscle. Intubating conditions were compared between both groups by means of a standardised score (the Copenhagen score) with Fisher’s exact test. ABSTRACT.RESULTS: Onset of the block after rocuronium injection was observed at the adductor pollicis muscle compared to the trapezius with 2.8 (1.1) versus 2.5 (1.1) min (mean ± SD; P = 0.006). Intubating conditions were poor in 2 patients (7%) of group A, and in 1 patient (3%) of group T. They were acceptable (either excellent or good) in 28 patients (93%) in group A, and in 1 patient (97%) in group T (P = 0.82). ABSTRACT.CONCLUSIONS: Performing acceleromyography at the trapezius muscle reduced the time between injection of neuromuscular blocking agents and intubation by 18 s (11%). Thus, trapezius muscle acceleromyography is an acceptable alternative to adductor pollicis muscle acceleromyography in predicting acceptable intubating conditions, which allows for earlier indication of adequate intubating conditions. ABSTRACT.TRIAL REGISTRATION: ClinicalTrial.gov Identifier: NCT01849198. Registered April 29, 2013. BODY.BACKGROUND: An adequate neuromuscular block improves intubating conditions and increases the rate of successful intubation. Thus, the risks of laryngeal injuries or pulmonary aspiration are reduced [1]. Unfortunately, neuromuscular measurements at the adductor pollicis muscle, probably the location used most frequently in the clinical routine, correlate poorly with intubating conditions. The reason for this observation might be that onset is slower and resistance to neuromuscular blocking agents is less compared to diaphragm or larynx [2–4]. Acceleromyography of the adductor pollicis muscle is commonly used to assess for adequate neuromuscular blockade. However, the onset of a neuromuscular block varies considerably depending on the muscle group used for assessment: for example, compared to the adductor pollicis muscle, a faster onset could be observed at the larynx [4], diaphragm, or masseter muscle [5, 6]. Correlation between acceleromyography and intubating conditions were better with the masseter than with the other muscles. Therefore, the ability to predict maximum neuromuscular block with acceleromyography depends on the muscle site used for acceleromyographic assessment. In this study, a recently introduced location for acceleromyography – the trapezius muscle - was examined and compared to a simultaneous stimulation of the adductor pollicis muscle [7]. Onset and recovery at the trapezius muscle were faster than at the adductor pollicis muscle. Therefore, the present study was performed to compare these two muscles with regard to prediction of good intubating conditions. Our hypothesis was that the trapezius muscle might reflect the laryngeal muscles better because of its proximal localization and might therefore be an alternative to the adductor pollicis muscle. The primary outcome was the percentage of good or excellent intubating conditions at both measurement sites. BODY.METHODS: BODY.PATIENT SELECTION: After approval of the local ethics committee and having obtained written informed consent (Ethikkommission der Ärztekammer Nordrhein, Duesseldorf, Germany, April 23th, 2013; No 2013056), we performed this prospective, unblinded, single center, randomized and controlled study (ClinicalTrial.gov Identifier: NCT01849198). We recruited female patients from 18 to 65 years, American Society of Anesthesiologists physical status I-III, body weight 50 to 90 kg, undergoing elective laparoscopic gynecological surgery. Exclusion criteria were: expected difficult tracheal intubation (e.g. history of difficult intubation, reduced opening of the mouth (< 2 cm) and/or Mallampati Score 4), increased risk of pulmonary aspiration (e.g. gastroesophageal reflux or delayed gastric emptying) known allergies to drugs administered during the study, pregnancy, neuromuscular diseases, medication with potential to influence the neuromuscular function (e.g., furosemide, magnesium or cephalosporins) and hepatic or renal insufficiency (serum bilirubin >26 μmol/L, serum creatinine >90 μmol/l). BODY.ANESTHESIA: All patients were pre-medicated with midazolam 7.5 mg per os. In the operating room, they received 100% oxygen via facemask together with an intravenous infusion of remifentanil 0.2 μg kg−1 min−1. Induction of anesthesia was performed with fentanyl 2 μg/kg and propofol 2–3 mg/kg. Initially a laryngeal mask (Ambu® AuraOnce™ size 4, Ambu Inc., Glen Burnie, MD 21060 USA) was inserted and anesthesia was maintained by continuous infusion of remifentanil 0.15–0.25 μg/kg/min and propofol 3–5 mg/kg/h. Eventually the laryngeal mask was replaced by an endotracheal tube (Rueschelit® 7.0 mm I.D., TeleflexMedical, Athlone, Ireland) after onset of the neuromuscular block. Patients’ lungs were ventilated with a respiratory rate of 10–12/min and a tidal volume of 6–8 ml/kg in order to achieve normocapnia, defined as an end-tidal CO2 concentration of 36–40 mmHg. Hemodynamic parameters were maintained within ±20% of baseline values. After placement of the laryngeal mask, neuromuscular transmission was measured by simultaneous monitoring of acceleromyographic responses (TOF Watch SX, Essex Pharma GmbH, Munich, Germany) at the adductor pollicis muscle and the trapezius muscle. Both muscles were stimulated using transcutaneous Ag/AgCl electrodes (electrocardiogram electrodes; Ambu Inc., MD 21060 USA) placed at the wrist of the right hand, and 1 cm dorsal to the inferior border of the left sternocleidomastoid muscle, respectively (Fig. 1). The right hand was fixed while the thumb was free to move [8]. Thus, contractions of the hand influencing the measurements at the thumb during ulnar stimulation were avoided. With regard to the accessory nerve, adequate stimulation was verified by measuring the movements of the left shoulder in a cranial direction.Fig. 1Position of the electrodes at the accessory nerve and the piezoelectric element (white arrow) on the trapezius muscle. Blue lines: sternocleidomastoid muscle. Red line: anterior border of the trapezius muscle. Yellow lines: accessory nerve The accelerometers’ piezoelectric elements were fixed distally at the right thumb [8], and at the left trapezius muscle in a distance of approximately 10 cm from the electrodes (Fig. 1). Both accelerometer were calibrated to deliver a supramaximal train of four (TOF) stimulus (0.2 Hz every 15 s, duration 0.1 ms). The TOF Watch SX automatically determines the individual supramaximal stimulation current (up to a maximum current of 60 mA). These maximal acceleromygraphic responses served as control values [9]. The first of the four twitch height responses was regarded to be T1, and the TOF ratio was calculated as the ratio of the fourth twitch (T4) height response and T1. During the first minutes of the acceleromyography, the electric current of the stimulation often changes the impedance of the electrodes thereby causing a drift of the acceleromyographic responses. Therefore, both acceleromyographs were recalibrated 10 min after start of the first stimulation. After a constant signal had been established at both measurement sites, rocuronium 0.3 mg/kg iv was injected over a period of 5 s. Directly afterwards, the intravenous line was flushed with Lactated Ringers’ solution, and neuromuscular block height was measured simultaneously at both locations every 15 s. The obtained data were stored on 2 computers which were connected to the TOF Watch SX devices (TOF-Watch SX Monitor Version 2.5.INT; Organon Ltd., Dublin, Ireland). Onset time and maximum block height were obtained as recommended by Fuchs-Buder et al. [8]. Onset time was defined as follows: Time from start of injection of rocuronium until T1 height fell <5% compared to baseline values. In case of an incomplete block (minimal T1 height 5% or higher compared to baseline values), onset time was measured as the time from start of injection of rocuronium until at least 3 consecutive twitches with the same or even increasing amplitude were observed. The first of these T1 twitches served as endpoint for calculation of the onset time (Fig. 2).Fig. 2Measurement of onset time in case of an incomplete neuromuscular block (defined as T1 height did not fall <5% of baseline values): in case of an incomplete onset time was measured as the time from start of injection of rocuronium until at least 3 consecutive twitches with the same or even increasing amplitude were observed. The first of these T1 twitches served as endpoint for calculation of the onset time (red arrow and black vertical line, respectively) Maximum block height was regarded as height of T1 at the onset of the block in percent compared to baseline values. BODY.ASSESSMENT OF INTUBATING CONDITIONS: Patients were randomized to two groups with different time points of endotracheal intubation according to a computerized allocation schedule: in group A (n = 30), intubation was performed when onset time was reached at the adductor pollicis muscle. In group T (n = 30), intubation was performed when onset time was reached at the trapezius muscle. Patients’ tracheas were always intubated by the same experienced anesthetist. Intubating conditions were assessed by means of the following variables [8]: ease of laryngoscopy (laryngoscopy component), position and movement of the vocal cords (vocal cord component), coughing or movement of the limbs during or directly after intubation (reaction to intubation component). Each of these variables could be rated as excellent, good or poor (Table 1). Intubating conditions were rated as excellent if all variables were excellent, they were good if all variables were good or excellent, and they were poor if any variable was poor.Table 1Criteria used to assess intubating conditions. Overall intubating conditions were rated as excellent if all variables were excellent, they were good if all variables were good or excellent, and they were poor if any variable was poor [8] Variable assessedClinically acceptableClinically not acceptableExcellentGoodPoorLaryngoscopy Easy Fair Difficult vocal cords position Abducted Intermediate/moving Closed Reaction to insertion of the tracheal tube and cuff inflation (diaphragmatic movements/coughing) none slight Vigorous/sustained Laryngoscopy: easy: jaw relaxed, no resistance to blade insertion. Fair: jaw not fully relaxed, slight resistance to blade insertion. Difficult: poor jaw relaxation, active resistance of the patient to laryngoscopy. Reaction to insertion of the tube: slight: one or two movements for less than 5 s. Vigorous/sustained: more than 2 contractions/movements for longer than 5 s BODY.STATISTICAL ANALYSIS: Statistical analysis was performed with Sigma Plot 12.3 for Windows software package (Systat Software Inc., Chicago, IL). Maximum block height and onset time of neuromuscular block were analysed with a paired t-test, because both muscles could be monitored in the same patient. Comparison between the groups were performed with an unpaired t-test (demographic data).Non-parametric data were analysed with Fisher’s exact test (intubating conditions). The primary outcome parameter was assessed with a subjective ranking scale. Therefore, a 33% difference between groups with regard to excellent or good intubating conditions was regarded as clinically relevant. To detect this difference with P < 0.05 and a power of 80%, 29 patients per group were required. BODY.RESULTS: Sixty patients were included into the study between June 2013 and May 2016 (Fig. 3). The demographic data did not differ between groups and are displayed in Table 2.Fig. 3Flow chart of patient selection. Group A: measurement of intubating conditions when onset time was reached at the aductor pollicis muscle. Group T: measurement when onset was reached at the trapezius muscle Table 2Data of the patients Group A (n = 30)Group T(n = 30) P valueage (years) 41 (8.1) 41 (6.8) 0.918 weight (kg) 67.4 (8.7) 68.7 (10.9) 0.624 height (cm) 166 (6.4) 167 (4.7) 0.715 BMI (kg/m2) 24.5 (3.0) 24.7 (3.9) 0.79 ASA 1.5 (0.5) 1.4 (0.5) 0.38 Data are presented as mean (SD). Group A: measurement of intubating conditions after onset of the neuromuscular block at the adductor pollicis muscle. Group T: measurement after onset at the trapezius muscle. ASA: American society of Anesthesiologists physical status BODY.ONSET TIME AND MAXIMAL NEUROMUSCULAR BLOCK: The supramaximal stimulation currents required for the measurements were similar in both groups with 49.2 ± 11.6 mA in group A, and 49.8 ± 11.6 mA in group T, respectively (mean ± SD). Following injection of rocuronium, maximum block was recorded slightly later at the adductor pollicis muscle compared to the trapezius muscle: 2.8 ± 1.1 versus 2.5 ± 1.1 min, respectively, (mean ± SD; P = 0.006). The corresponding T1 twitch heights recorded did not differ: 2.8 ± 5.2% at the adductor pollicis muscle versus 1.9 ± 5.2% at the trapezius muscle (mean ± SD). Data are displayed in Table 3.Table 3Course of the neuromuscular block adductor muscle (n = 60)trapezius muscle (n = 60) P valuesupramaximal stimulation (mA) 49,2 (11.6) 49.8 (11.6) 0.77 onset time (min) 2.8 (1.1) 2.5 (1.1)* 0.006 twitch height (% T1 height) 2.8 (5.2) 1.9 (5.2) 0.33 Data are presented as mean (SD). Adductor muscle: neuromuscular measurements obtained at the adductor pollicis muscle; trapezius muscle: measurements obtained at the trapezius muscle. Supramaximal stimulation: strength of supramaximal stimulation in mA; onset time: time between the beginning of injection of rocuronium and maximum T1 depression; twitch height: twitch height in percent compared to baseline values; n: number of successful measurements. *: P < 0.05 for trapezius vs. adductor pollicis BODY.INTUBATING CONDITIONS: Tracheal intubation was performed without complications or difficulties in all patients of both groups. Overall intubating conditions were poor in 2 patients (7%) of group A, and in 1 patient (3%) of group T. They were acceptable (excellent or good) in 28 (93%) of the patients in group A, and in 29 (97%) of the patients in group T (P = 0.82). Thus, differences with regard to intubating conditions were not observed between groups. No differences were found between groups with regard to the 3 assessed variables (laryngoscopy, vocal cords and diaphragm). Details are provided in Table 4.Table 4Intubating conditions when onset time of the neuromuscular block was reached at the adductor pollicis muscle or the trapezius muscle, respectively [8]. No significant differences between groups Intubating conditionsGroup A (n = 30)Group T (n = 30) P valueLaryngoscopy: 0.07  easy 25 30  fair 4 0  difficult 1 0 Vocal cords: 0.73  abducted 26 24  intermediate 4 6  closed 0 0 Diaphragmatic movements: 1.0  none 26 26  slight 3 3  sustained 1 1 Overall intubating conditions 0.82  excellent 22 22  good 6 7  poor 2 1 Group A: measurement of intubating conditions after onset of the neuromuscular block at the adductor pollicis muscle. Group T: measurement after onset at the trapezius muscle BODY.DISCUSSION: The present study compared the intubating conditions at two different acceleromyographic measurement sites (adductor pollicis and trapezius muscles) after rocuronium administration. Intubating conditions were assessed by the Copenhagen Scoring System. This score standardises the observations in three categories thereby facilitating the data comparison from different studies. Therefore, its use is recommended for studies assessing intubating conditions [8]. Although the onset times differed from each other at the two measurement sites, intubating conditions were similar in both groups. Several studies have been performed to find a more suitable stimulation site in order to predict optimal intubating conditions. Lee et al. performed a study assessing intubating conditions at maximal neuromuscular block at 3 different sites: the adductor pollicis muscle, the orbicularis oculi muscle and the corrugator supercilii muscle [10]. Twitch observation at the orbicularis oculi muscle allowed a faster intubation. Unfortunately, the frequency of inadequate conditions was increased. The authors observed best intubating conditions if the adductor pollicis muscle was chosen to predict onset of the neuromuscular block; however, with the longest delay between start of anesthesia and tracheal intubation. In a second study, they compared orbicularis oculi, corrugator supercilii, masseter and mylohyoid muscles with each other [11]. Again, best intubating conditions were observed at the measurement sites with the longest delay between induction and complete neuromuscular block (mylohyoid muscle). The authors concluded that monitoring of the corrugator supercilii muscle provided the best compromise between acceptable intubating conditions and short onset. This statement is supported by the findings of Plaud et al. observing that the corrugator supercilii muscle reflected onset of the block at the laryngeal muscles better than the orbicularis oculi muscle [12]. However, the movements of the orbicularis oculi and the corrugator supercilii muscles were small; therefore, quantifying the measurements was difficult [12]. Finally, these muscles lie close to the corresponding facial nerves. Therefore, the risk of accidental direct stimulation of the muscle instead of the nerve is relatively high. Conversely, other authors found that monitoring onset of the neuromuscular block at the orbicularis oculi muscle would be able to predict good intubating conditions [13]. In the light of these observations, acceleromyography at the trapezius muscle might have several advantages: it is as easy to perform as e.g. measurement at the adductor pollicis muscle, it produces reliable data, and there is no need of specialized equipment [7]. Moreover, manipulations such as mask ventilation do not interfere with assessment to the same degree as with measurements at the orbicularis oculi muscle and the corrugator supercilii muscles. Kitajima et al. compared tactile and acceleromyographic assessment of the masseter muscle with acceleromyography of the adductor pollicis muscle [5, 6]. They observed a faster onset at the masseter muscle with intubating conditions graded as good or excellent. The masseter muscle is localised near to the trapezius muscle. Therefore, these observations are consistent with the results of our investigation demonstrating similar results with a faster onset at the trapezius muscle without observing deteriorated intubating conditions. Taken together, the literature is equivocal with regard to the optimal measurement site for assessment of good intubating conditions. Thus, further research addressing this topic would be of interest. Studies assessing intubating conditions are methodologically difficult to perform, because it is difficult to quantify the evaluation. In our study we adhered to the recommendations for studies assessing neuromuscular blocking agents [8]. We evaluated intubating conditions by the Copenhagen score introduced by Viby-Mogensen [14] in order to make our results comparable to the data of other authors. In addition, we used a relatively low dose of rocuronium making it easier to detect small differences between the groups. The depth of anesthesia is known to influence intubating conditions: therefore, all variables of anesthesia were standardised. The size of the groups was similar to those in other studie10, 11, 15], and the rate of excellent intubating conditions (73%) was comparable to the results of other authors observing 70 to 80% [16, 17]. However, we were not able to find differences between both measurement sites with regard to intubating conditions. A reason for this result might be that most patients had maximum neuromuscular blocks in spite of low dose of rocuronium in both groups. Thus, the high percentage of acceptable conditions is not surprising. Another limitation might be the restriction on female patients. Women usually require lower doses of neuromuscular blocking agents than men. Therefore, our approach made the groups more homogenous. On the other hand, the inclusion of male patients might have increased the rate of inadequate intubating conditions because of an insufficient neuromuscular block. BODY.CONCLUSIONS: Performing acceleromyography at the trapezius muscle reduced the time interval between induction of anesthesia and tracheal intubation by 18 s (11%). Thus, trapezius muscle acceleromyography is an acceptable alternative to adductor pollicis muscle acceleromyography in predicting acceptable intubating conditions, which allows for earlier indication of adequate intubating conditions.

TITLE: Acupuncture for dyspnea in advanced cancer: a randomized, placebo-controlled pilot trial [ISRCTN89462491] ABSTRACT.BACKGROUND: Dyspnea, or shortness of breath, is a common symptom in patients with advanced cancer. Pharmacologic management is of proven benefit, but it does not help all patients. Preliminary data suggest that acupuncture can relieve dyspnea in a variety of populations, including cancer patients. We conducted a pilot study (ISRCTN89462491) preparatory to a fully powered randomized, placebo-controlled trial to determine whether acupuncture reduces dyspnea in patients with lung or breast cancer. ABSTRACT.METHODS: The study sample was comprised of forty-seven patients with lung or breast cancer presenting with dyspnea. Patients receiving symptomatic treatments were not excluded as long as no changes in management were planned during the trial. Patients were randomized to receive a single session of true or placebo acupuncture in addition to their existing dyspnea treatments. Semi-permanent acupuncture "studs" were then inserted: patients applied pressure to these studs twice a day to provide ongoing stimulation to acupuncture points. The subjective sensation of dyspnea was assessed with a 0 – 10 numerical rating scale immediately before and after acupuncture treatment and daily for a week thereafter. ABSTRACT.RESULTS: All but two of 47 randomized patients provided follow-up data. Dyspnea scores were slightly higher for patients receiving true versus placebo acupuncture, for both the period immediately following acupuncture treatment and for the daily one week follow-up (differences between means of 0.34, 95% C.I. -0.33, 1.02 and 0.56, 95% C.I. -0.39, 1.51). The 95% confidence interval excludes the prespecified minimum clinically significant difference of a 20% greater improvement in dyspnea for patients receiving acupuncture. ABSTRACT.CONCLUSION: The acupuncture technique used in this trial is unlikely to have effects on dyspnea importantly larger than placebo for patients with advanced cancer. BODY.BACKGROUND: Dyspnea, or shortness of breath, is defined as the subjective experience of difficulty breathing. It is a common symptom in cancer patients, particularly those with advanced cancer. For example, in a general survey of 100 outpatients and 140 inpatients at a Veterans' Affairs Medical Center, at least some dyspnea was reported by 50% of patients [1]. Studies of patients with advanced disease find that 50 –70% experience significant dyspnea[2,3]. Although dyspnea is most commonly associated with lung cancer[3], high rates also occur in patients with breast cancer with pulmonary metastases[4]. Dyspnea in cancer patients has numerous causes. It may result from complications of the cancer itself, such as pulmonary embolism, pleural effusion, anemia, and endobronchial obstruction or from conditions associated with a risk factor such as chronic obstructive pulmonary disease (COPD) in smokers. It may also result from disorders not directly attributable to the lungs, such as congestive heart failure or anemia. Treatment of these patients is typically guided by the identification of a specific underlying abnormality. Pleural effusions, for instance, are treated by thoracentesis, COPD is treated by bronchodilators and/or steroids, and anemia is treated by transfusion. Unfortunately, a specific lung or cardiac pathology is not identifiable in approximately one-quarter of patients[2]. Moreover, dyspnea may result from causes in which definitive treatment has been unsuccessful or not practical. A number of interventions have been attempted for such patients, who often have advanced cancer within the lung parenchyma or endobronchial disease refractory to external beam radiation. Several randomized trials have shown that both opioids and supplemental oxygen can alleviate the subjective sensation of dyspnea among patients with advanced cancer. For example, in a randomized, double-blind, crossover trial, dyspnea sensation fell by 25 mm on a 100 mm visual analog scale after morphine, with little change on placebo (p < 0.01 for difference between groups)[5]. Using a similar randomized, crossover design, Bruera et al. reported highly significant differences between periods when patients received oxygen as compared to periods when unsupplemented air was administered [6]. However, such measures are not effective in all patients, and the problem of dyspnea in the cancer patient is often frustratingly difficult to treat. A multinational study of end-of life-care found that dyspnea required sedation in 25 to 50% of patients during the last week of life[7]. It is for these difficult to treat cancer patients that additional therapeutic options are needed. A number of randomized controlled trials have examined acupuncture for shortness of breath in patients without cancer. For example, a randomized, double-blind, placebo-controlled trial of acupuncture for methacholine-induced asthma reported significant differences in favor of acupuncture[8]. A similar study involved a standardized running test to induce bronchorestriction in asthmatics. Exercise-induced reductions in lung function were significantly lower in real compared to placebo acupuncture[9]. A trial in COPD reported significant differences between groups for subjective breathlessness. Thirty-one patients were randomized to self-administer finger pressure ("acupressure") at true or sham acupuncture points on a double-blind, crossover basis. Acupressure led to approximately a one-third reduction of scores on a VAS of dyspnea compared to about a 20% improvement in placebo controls[10]. Only one trial has been reported in cancer. Although uncontrolled, the results are provocative. Thirty cancer patients in palliative care received a single session of acupuncture. The mean visual analog score for breathlessness before acupuncture treatment was 42; this fell to 24 immediately following 10 minutes of needle insertion, an improvement that was maintained at six hour follow-up. Symptom scores returned to baseline 24 hours later[11]. Given these data, we believed it would be worthwhile to investigate whether cancer-related dyspnea can be relieved by acupuncture. Acupuncture is a complementary therapy, and therefore was administered as a supplement to use of pharmacologics or oxygen for dyspnea. Placebo control was employed because our endpoint was subjective and because acupuncture had not previously been found superior to placebo for cancer-related dyspnea. Our overall objective was to determine whether true acupuncture was more effective than placebo for alleviating dyspnea in patients with advanced lung or breast cancer. Here we report the results of a pilot study that aimed to refine the methodology for an anticipated definitive trial. BODY.METHODS: Recruitment for the trial took place between July 2001 and March 2004 among patients under treatment for advanced lung or breast cancer at Memorial Sloan-Kettering Cancer Center (MSKCC). Patients age 18 or above with subjective complaint of shortness of breath and scoring grade two or higher on the American Thoracic Society Breathlessness Scale were eligible. Patients must have pursued a trial of steroid medication for dyspnea, if indicated, for at least 48 hours. Patients were excluded if any of the following applied: shortness of breath predated cancer diagnosis (e.g. asthma); recent onset of symptoms (< 7 days); anemia (defined as hemoglobin < 8 gm/dl); recent acupuncture; contraindications to acupuncture such as heart valve dysfunction or pancytopenia; planned initiation or change in oncologic therapy or symptomatic management of breathlessness (continuation of any existing management was allowed), or likelihood of patient death during the course of anticipated participation in the trial. Patients were also excluded if the primary cause of dyspnea was thought to be congestive heart failure, sarcoid disease, hypersensitivity pneumonitis, cryptogenic organizing pneumonitis, pneumothorax, chest wall deformity, obesity, neuromuscular disorders, pulmonary vascular disease, hepatomegaly or phrenic nerve paralysis syndrome. If the primary cause of dyspnea was ascites, effusion, pneumonia, large airway obstruction, superior vena cava syndrome or pulmonary embolism, patients were eligible only if they remained short of breath despite conventional therapy administered by their primary oncologist, or if they had refused such therapy. Eligible consenting patients were randomized by telephone using the MSKCC clinical research database. Randomization used randomly permuted blocks with the cancer diagnosis (lung/breast) and breathless at rest (yes/no) as strata. The use of independent telephone registration and randomization ensured concealment of treatment allocation. Patients, researchers and others involved in patient care were blind to study group; only the acupuncturists and a researcher not associated with the study were aware of which patients received true and which placebo treatment. Treatment consisted of two phases, acupuncture and acupressure. Patients received either true acupuncture followed by true acupressure, or placebo acupuncture followed by placebo acupressure. The first "acupuncture phase" consisted of a single treatment in which real or placebo needles were applied for 15 minutes at true or sham points, respectively. In the true acupuncture group, the needles used were stainless steel AsiaMed No.16 30 mm × 0.30 mm inserted to the traditional Chinese depth of 0.5 cm – 1.5 cm. Auricular points were needled with stainless steel Seirin D type No. 1 15 mm × 0.16 mm. As is common in traditional acupuncture, practitioners attempted to elicit de qi to help determine exact point location, but there was no manipulation of needles after placement. Placebo needles consist of a blunted needle that moves up inside its handle instead of into the skin. This technique has previously been demonstrated to be indistinguishable from true acupuncture[12]. Patients were told that in placebo acupuncture "the needles are placed so that they do not stimulate the correct acupuncture points. However, they do not look or feel any different from real needles." Immediately after insertion of the needles, patients were asked to assess the credibility of their treatment using a previously published scale[13,14]. The point prescription is described in Table 1. True points were chosen on the basis of the prior case series[11] and points traditionally used for breathlessness[15]; sham points were chosen in body areas away from true acupuncture points. The point prescription was modified slightly during the trial (details available on request), primarily to improve patient comfort. Patient outcome was not affected by the prescription used (see Results). Immediately after needle insertion, acupuncturists completed an audit sheet verifying the acupuncture points used. These records were routinely reviewed, and acupuncturists were found to have followed the acupuncture point prescription. Table 1Location of acupuncture points. Point             Laterality     Anatomical location Ren6 Unilateral On the midline of the lower abdomen, 1.5 cun* inferior to the umbilicus and 3.5 cun* superior to the pubic symphysis LU1 Bilateral On the lateral aspect of the chest, in the first intercostal space, 6 cun* lateral to the midline, LU7 Bilateral On the radial aspect of the forearm, in the cleft between the tendons of brachioradialis and abductor pollicis longus ST36 Bilateral Below the knee, 3 cun* inferior to Dubi ST35, one finger breadth lateral to the anterior crest of the tibia. KI6 Bilateral 1 cun* below the prominence of the medial malleolus, in the groove formed by two ligamentous bundles. Auricular lung point Bilateral Medial and distal aspect of the tragus Auricular kidney point Bilateral Medial and proximal aspect of the cymba conchae Sternal points OR Ren17 Unilateral Two needles inserted in the top two inches of the sternum and inserted down to the periosteum. Weak or cachectic patients were treated at Ren17, which is found on the anterior midline, at the level with the fourth intercostal space, midway between the nipples Sham sternal Unilateral Two points anywhere in the upper two inches of the sternum: placebo needle only Sham1 Bilateral Posterior wrist, between radius & ulna, 1 cun* distal to radial & ulnar heads Sham2 Bilateral Anterior arm, 3 cun* proximal and 3 cun* medial to the antecubital crease Sham3 Bilateral Anterior arm, center of biceps brachii (midway between shoulder joint and antecubital crease) Ear Sham Bilateral Adjacent to the "finger point", just below the helix of the auricle, at the apex * a "cun" is a practical measurement used by acupuncturists equivalent to the greatest width of a patient's thumb at the distal phalanx One hour after removal of needles, patients started the second "acupressure phase." Stainless steel AcuMedic acupressure studs, sometimes described as "press," "semi-permanent" or "intradermal" acupuncture needles, were used. These consist of a 2 mm × 0.28 mm acupuncture needle attached to a metal ring embedded in surgical tape. When the surgical tape is pressed on the skin, the needle pierces the skin but its diameter is so fine that this sensation is neither painful nor even immediately obvious. Acupressure studs have been used in a number of studies with cancer patients[16,17]. Placebo studs have no needle: they were specially designed for research in end-of-life populations by the author of the previous research on acupuncture for cancer related breathlessness[11]. To ensure retention of studs and guard against infection, the studs were covered with Tegaderm following application. Patients in the true acupuncture group were treated with true studs at ST36; Sternal points or Ren17; Auricular lung point and Auricular kidney point (see Table 1). Placebo patients received placebo studs at sham1, sham2 and ear sham. We thought it possible that patients might remove the studs before the end of the trial and that studs with a visible needle might be more credible than those without. Therefore, we also applied a placebo stud at sham1 to the acupuncture group and a true stud at sham3 to the placebo group. We told patients that some studs had small needles, others did not and that the choice depended on where they were placed on the body. Patients were also told that those in the placebo group received treatment at points not thought to help breathlessness. Following application of the studs, patients were instructed to apply pressure to the study by making small circular movements with the fingers of the opposite hand, 2 – 3 cycles per second for 1 – 2 minutes per point. As is typical for self-administered acupressure, patients were encouraged to apply acupressure this way on waking, in the early afternoon and during any exacerbation of symptoms. Initial instruction was provided verbally, at which time patients were asked to confirm their understanding by demonstrating the procedure. Patients also were given easy-to-read written materials describing the acupressure procedure. Study acupuncturists are certified by the National Certification Commission for Acupuncture and Oriental Medicine (NCCAOM) and are licensed to practice acupuncture in New York State. They have used acupuncture in clinical practice for 3 – 25 years. All were employed at MSKCC during the study and had considerable experience in treating cancer patients. Outcome was measured in two ways. Every 15 minutes for 75 minutes immediately before acupuncture treatment and one hour immediately after, patients rated their current level of breathlessness on a 0 to 10 rating scale. They then completed a dairy daily for seven days, recording their average level of breathlessness through the day with the same 0 to 10 scale, and recorded compliance with acupressure. The protocol was modified after 16 patients were accrued, as we found that many in-patients had been excluded because changes in management were likely in the subsequent seven days. We therefore modified the protocol so that inpatients did not take part in the acupressure phase. Moreover, many outpatients who complained of breathlessness in everyday life did not record significant breathlessness during the waiting period immediately before acupuncture. Accordingly, we amended the protocol so that outpatients completed the daily breathlessness diary for a week at baseline. Only patients scoring a mean of two or more were eligible. Out-patients who reported no breathlessness at rest were not asked to report on symptoms in the period immediately before and after acupuncture. Preliminary power calculations on the basis of published data[10,11] suggested that a sample of 120–150 patients would be required to provide power to detect a clinically significant difference between groups, defined as a 20% lower follow-up dyspnea score in the acupuncture group compared to placebo. For this pilot we sought a sample of 40–50 patients. We felt that a sample of this size would give us sufficient methodologic experience to conduct an adequate fully powered study. Comparisons between groups were by analysis of covariance (ANCOVA) with baseline score and randomization strata as covariates. Baseline score for the acupressure phase was the one week baseline diary, if available, otherwise the immediate pre-treatment breathlessness score was used. Patients were analyzed in their randomized groups regardless of treatment received. Prespecified sensitivity analyses were to examine the effects of credibility on outcome and to assess outcome for the acupuncture phase only in patients with pre-treatment dyspnea scores greater than two. Statistical analysis was conducted using Stata 8 (College Station, Texas). No interim analyses were planned and the data were not analyzed before study closure. The study was approved by the institutional review board at MSKCC in accordance with an assurance filed with and approved by the Department of Health and Human Services. Written informed consent was obtained from each participant. BODY.RESULTS: Flow of participants through the trial is shown in figure 1. Raw data are provided in Additional file 1. Table 2 shows data on study completers. Groups are balanced for baseline characteristics such as age, sex and diagnosis, and for postrandomization characteristics such as credibility and compliance. The similarity among credibility scores suggests that blinding was maintained, a conclusion also supported by analysis of research assistant notes: of the 21 patients who commented on allocation during post-study debriefing, 12 claimed to be unaware of allocation and only four of the remaining nine made a correct guess as to treatment received. Use of steroids, slightly more common in the placebo group, was not a strong predictor of outcome and there was no interaction between steroid use and group allocation. No patient changed use or dose of diuretics, opiates, bronchodilators or steroids during study participation. Figure 1Flow of participants through the trial. Table 2Baseline characteristics of patients providing post randomization data. Values are number (percentage) or mean (standard deviation) Acupuncture n = 25 Placebo n = 20 Female 15 (60%) 13 (65%) Age 63.0 (12.8) 67.0 (11.4) Diagnosis  breast 5 (20%) 4 (20%)  lung 20 (80%) 16 (80%) Breathless at rest 9 (36%) 6 (30%) Credibility score* 2.24 (0.46) 2.10 (0.59) Steroid use:**  None 15 (60%) 9 (47%)  <40 mg prednisone equivalents 6 (24%) 8 (42%)  40+ mg prednisone equivalents 4 (16%) 2 (11%) Diuretics** 3 (12%) 0 (0%) Opiates** 5 (20%) 3 (16%) Bronchodilators** 12 (48%) 7 (37%) Fully compliant with acupressure+ 8 (80%)° 6 (75%)°° * two missing observations in acupuncture group; three missing observations in placebo group ** one missing observation in placebo group + n = 10 acupuncture, n = 8 placebo ° one patient completely non-compliant, one patient 85% compliant °° one patient 33% compliant, one patient 85% compliant Breathlessness scores are reported in Table 3. We give results separately for dyspnea measured immediately after acupuncture, and for dyspnea assessed by daily diary. Some patients reporting dyspnea scores immediately after acupuncture treatment had negligible pre-treatment dyspnea. Hence we also provide a sub-group analysis of the immediate post-treatment scores, excluding patients scoring a mean of 2 or below. For all analyses, patients receiving true acupuncture reported slightly higher scores at follow-up, approximately equivalent to 10% of baseline. Patients in both groups improved, but no important differences between groups emerged. The upper bound of the 95% confidence interval for the difference between means excludes our prespecified minimum clinically significant difference of a 20% greater improvement in dyspnea for patients receiving acupuncture. Credibility did not predict breathlessness scores, either in the immediate post-treatment period (p = 0.4) or during the one week of diary recording (p = 0.9). Moreover, including credibility in the model for treatment effect did not importantly modify estimates. Table 3Breathlessness scores. Baseline scores only given for patients providing relevant follow-up data. Values are mean (standard deviation) Endpoint Group Baseline Follow-up p value for within group change Difference between means+ 95% C.I. p value for between group comparison Immediately post-treatment Acupuncture n = 19: 4.09 (2.32) n = 19: 3.36 (2.21) 0.003 0.34 -0.33, 1.02 0.3 Placebo n = 14: 3.41 (2.79) n = 14: 2.42 (2.64) 0.003 Immediately post-treatment (pre-treatment score >2) Acupuncture n = 15: 4.87 (1.92) n = 15: 3.99 (2.03) 0.003 0.45 -0.55, 1.46 0.4 Placebo n = 8: 5.28 (2.18) n = 8: 3.92 (2.50) 0.01 Mean of seven day breathlessness diary Acupuncture n = 10*: 6.58 (1.71) n = 16*: 5.07 (2.12) 0.4 0.56** -0.39, 1.51 0.2 Placebo n = 7*: 5.99 (1.71) n = 14*: 3.77 (2.39) 0.07 + Positive indicates worse score in acupuncture group. **n = 13 for placebo group: one patient had no baseline data. We conducted exploratory analyses to determine whether acupuncture prescription affected outcome. Outcome did not differ by point prescription in either the subgroup analysis (Table 4) or by including prescription and prescription by group interaction in the ANCOVA model. Table 4Effect of acupuncture point prescription on outcome. Endpoint Point prescription n Difference between groups+ 95% C.I. Immediately post-treatment Initial 15 -0.23 -1.23, 0.77 Modified 18 0.87 -0.19, 1.94 Daily Breathlessness diary Initial 12 0.63 -0.54, 1.79 Modified 17 0.43 -0.92, 1.77 + Positive indicates worse score in acupuncture group No adverse events were causally related to acupuncture treatment. BODY.DISCUSSION: Although intended as a methodologic pilot, our results are unexpectedly precise enough to warrant clinical recommendations. The confidence interval for the difference between groups is sufficiently narrow for us to conclude that, even in the best case scenario, the acupuncture technique tested is not importantly superior to placebo for dyspnea in cancer patients. Based on our clinical judgment of how much improvement we should expect to make worthwhile the time and trouble associated with acupuncture, we pre-specified a 20% difference between groups as clinically meaningful. It is against this criterion that we draw our conclusion of "no effect". Yet we can exclude even smaller levels of benefit, such as 10%. We find it unlikely that any patient or clinician would deem acupuncture to be worthwhile for a benefit of one third of a point on a 0 – 10 scale. There are several possible limitations of our study. First, protocol changes concerning intervention, eligibility and evaluation, although common for a methodologic pilot, are not ideal when drawing conclusions about the effectiveness of a treatment. Nonetheless, we feel that these protocol changes did not have an important influence on our results. Changes in the methods of evaluation were designed to reduce drop-out and improve the measurement of baseline symptoms. These changes should increase the precision of results. Amendments to eligibility requirements were made to ensure that all patients had non-zero symptom scores at baseline and thus ensure that the trial included only those for whom benefit was measurable. Modification of the intervention itself is potentially more problematic. Most of the acupuncture point prescription changes were made to increase patient comfort. For example, we initially used the PC6 point on the wrist, but patients complained that studs inserted there caused radiating pain, apparently due to the proximity of PC6 to the median nerve. If our initial, but not modified, prescription were of value, it would be clinically irrelevant as an effective but intolerable treatment has little clinical role. Moreover, we saw no evidence that outcome was affected by the particular prescription used. Patients in this study received only a single acupuncture treatment. This does not reflect traditional acupuncture practice, and it is possible that repeated treatment might have a cumulative effect. Our study design was based on prior research that suggested immediate effects on dyspnea following a single session of acupuncture[8,11] Delayed or cumulative effects are of questionable relevance for symptomatic management in patients with late stage cancer. Moreover, daily acupressure treatment had no apparent effect. It is possible that more encouraging findings may have emerged had we used a different acupuncture technique, such as modifying the point prescriptions for each patient. Our study population reflected a typical spectrum of cases requiring symptomatic management of dyspnea, and was thus rather heterogeneous. Therefore, the possibility that acupuncture might be effective for dyspnea in a sub-group of patients with a particular pathology cannot be ruled out. There is a dearth of systematically collected data to inform decisions about study design with respect to either the most effective acupuncture technique or the most responsive patient group. Our study provides some evidence of a placebo effect. Symptom scores fell by approximately 20% immediately after acupuncture in both groups (p = 0.003), a result unlikely to be explained by regression to the mean or other effects. It is plausible that such a result would have been considered "positive" in a single-arm study, underscoring the importance of randomized trials for evaluating complementary therapies used to treat cancer-related symptoms. Accrual to the study was relatively slow, with only approximately 15 patients entering per year. This is due in part to the fact that MSKCC does not provide hospice services, which limits the number of end of life patients, when dyspnea is most prevalent. Accrual was also limited by patient refusal, as nearly half of patients approached declined participation. Patients with advanced cancer admitted to MSKCC generally have severe medical problems. Accordingly, they often feel overwhelmed, and a clinical trial, regardless of its perceived benefits and risks, is perceived as an additional burden. Patients commonly noted in response to the invitation to participate that "I just can't face dealing with this right now". BODY.CONCLUSION: We conclude that the acupuncture technique used in this trial is unlikely to have effects importantly larger than placebo for dyspnea in patients with advanced cancer. BODY.ABBREVIATIONS: ANCOVA: analysis of covariance COPD: chronic obstructive pulmonary disease VAS: visual analog scale MSKCC: Memorial Sloan-Kettering Cancer Center BODY.COMPETING INTERESTS: The author(s) declare that they have no competing interests BODY.AUTHORS' CONTRIBUTIONS: AJV, BRC and MBF designed the study. AJV analyzed data and wrote the manuscript. GED gave input to the analysis and manuscript with respect to the interpretation of the findings. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: BODY.SUPPLEMENTARY MATERIAL: BODY.ADDITIONAL FILE 1: This is an Excel file with raw data from the study. The first worksheet contains the data; the second, a list of variable descriptors. Click here for file

TITLE: Effect of esomeprazole versus placebo on pulmonary exacerbations in cystic fibrosis ABSTRACT.BACKGROUND: Gastro esophageal reflux (GER) is common in cystic fibrosis (CF) and may contribute to lung disease. Approximately 50% of patients with cystic fibrosis are being treated with proton pump inhibitors (PPIs). ABSTRACT.METHODS: In a randomized controlled study in adults, we compared treatment with esomeprazole 40 mg twice daily versus placebo in patients with CF and frequent respiratory exacerbations over a thirty-six week treatment period to determine effect on time to first exacerbation and other health related outcomes. ABSTRACT.RESULTS: 17 patients without symptoms of GER were randomized and 15 completed the study. 13 subjects underwent 24 hour ambulatory pH probe monitoring; 62% had pH probe evidence of GER. Forty one percent of subjects had a pulmonary exacerbation during the study. There was no significant difference in time to first pulmonary exacerbation (log rank test p = 0.3169). Five of nine subjects in the esomeprazole group compared with 2 of eight subjects in the placebo group experienced exacerbations (esomeprazole vs. placebo: odds ratio = 3.455, 95% CI = (0.337, 54.294), Fisher’s exact test: p = 0.334). There was no change in Forced Expiratory Volume in one second, Gastroesophageal Symptom Assessment Score or CF Quality of Life score between the two treatment groups. ABSTRACT.CONCLUSIONS: There was a trend to earlier exacerbation and more frequent exacerbations in subjects randomized to esomeprazole compared with placebo. The effect of proton pump inhibitors on pulmonary exacerbations in CF warrants further investigation. ABSTRACT.CLINICAL TRIALS REGISTRATION: Clinicaltrials.gov, NCT01983774 BODY.BACKGROUND: Gastroesophageal reflux (GER), both symptomatic and silent, is frequent in patients with cystic fibrosis (CF), and is often regarded as playing a role in the pathogenesis of CF related lung disease [1-4]. The overall prevalence of GER in CF is not well established, but is reported to be as high as 80% when diagnosed by esophageal pH-probe monitor in CF adults [3,5]. One study reported that 91% of patients with CF awaiting lung transplant had evidence of GER by pH probe monitoring [6]. Symptoms of lung disease in CF may overlap with pulmonary symptoms of gastroesophageal reflux, making it difficult to distinguish between the two conditions and often leading to treatment of both conditions. In 2010 in the US, 48% of adults and 51% of children with CF were being treated with proton pump inhibitors [7]. Several studies have suggested that patients with CF who have GER have more severe lung disease with lower pulmonary function and increased numbers of respiratory exacerbations [2,8]. In a prospective study, Button etal demonstrated that children with CF receiving modified chest physiotherapy with avoidance of head in the tilt down position not only had reduced episodes of GER as measured by ambulatory pH probe, but also had reduced need for antibiotics, reduced number of hospital days and improved lung function over a five year period [9]. The European Epidemiologic CF Registry reported that patients with CF and GER had lower pulmonary function than those without GER [8]. A recently conducted retrospective study of Nissen fundoplication in patients with CF and GER showed a significant decline in pulmonary exacerbations and improvement in forced expiratory volume in one second (FEV1) during the two years following surgery compared to the two years preceding surgery [10]. Despite considerable evidence that GER is common in CF and may be associated with more severe lung disease, the effect of acid suppressor therapy on improving lung function and reducing pulmonary exacerbations has not been prospectively studied. Proton pump inhibitors (PPIs) suppress the production of gastric acid and several studies have tested their effectiveness in improving pulmonary outcomes in chronic respiratory diseases. Studies of PPI therapy in asthma have inconsistently demonstrated beneficial effects [11,12], and retrospective studies in idiopathic pulmonary fibrosis suggest stabilization of lung function and improved survival with acid suppression [13,14] , Among individuals with CF , PPIs are likely initiated for a variety of reasons including improved efficacy of pancreatic enzymes in a higher pH environment, as well as treatment of cough or other respiratory or gastrointestinal complaints thought to be possibly caused by GER. Use of these agents however, may be associated with risk [15,16]. Use of PPIs in both hospitalized and ambulatory patients has been shown to be associated with an increased risk of pneumonia [15-18]. Furthermore, PPIs have been implicated in accelerated bone loss [19,20]. We compared treatment with esomeprazole versus placebo in a pilot study of patients with CF and frequent respiratory exacerbations to determine whether suppression of gastric acid leads to longer time to first pulmonary exacerbation and improvements in other health related outcomes. BODY.METHODS: We conducted a randomized, placebo-controlled double blind trial of esomeprazole in adult patients with cystic fibrosis. Adults with cystic fibrosis were enrolled from the clinical practices of two adult cystic fibrosis programs in New York City. Inclusion criteria were age of 18 years or older and two to four respiratory exacerbations per year requiring oral and/or intravenous antibiotics for each of the two years prior to study entry. At the time of enrollment, subjects had to have been on a stable maintenance medical regimen for at least six weeks. Participants were excluded if they were being treated with PPIs, were receiving enteral feeds, had smoked cigarettes within the previous six months, had previous anti-reflux surgery or clinical indications for acid-suppressor treatment (i.e. two or more episodes per week of heartburn requiring antacids). Participants were also excluded if they were being treated with medications that interact with proton pump inhibitors (azoles, iron, anti-coagulants), were pregnant or had a pulmonary exacerbation requiring antibiotics within the previous two weeks. All participants provided written informed consent statements that had been approved by the Columbia University Institutional Review Board (IRB AAAC8262) and the Beth Israel Medical Center Institutional Review Board (IRB 074-10). After the screening visit, those subjects who met eligibility criteria were enrolled in a 2 week run-in period during which time they underwent 24-hour ambulatory pH probe monitoring. Calibrated pH probes were placed in the distal esophagus using esophageal manometry, 5 cm above the lower esophageal sphincter. Criteria for an acceptable study included total recording time of at least 16 hours, with at least one meal and 2 hours of recumbency. A study was considered positive for distal GER if the distal pH was less than 4 more than 5.8% total time, or more than 8.2% of upright time, or more than 3.5% of supine time [21,22]. Meal times were excluded in the analysis to avoid false-positive data. A single gastroenterologist at each of the two centers reviewed studies; study subjects and study investigators were blinded to the results. Fourteen days after screening, subjects were randomly assigned in a 1:1 ratio to receive either esomeprazole 40 mg twice daily or matching placebo, regardless of pH probe results. The Columbia University Research Pharmacy prepared study medication. At the randomization visit, baseline spirometry, CF related quality of Life (CFQ-R QOL) [23] and Gastroesophageal Symptom Assessment Score (GSAS) measuring number and severity of reflux symptoms [24] were collected. Randomization was stratified based on study center and FEV1 decile. Primary outcome measure was time to first pulmonary exacerbation. Secondary outcomes included exacerbation rate, change in FEV1, forced vital capacity (FVC), CFQ-R QOL score and GSAS score. After randomization, participants returned to the clinic every six weeks for 36 weeks. Outcome measures were re-assessed at 12, 24 and 36 weeks after randomization. Subjects were instructed to notify the study site if they had signs or symptoms of a pulmonary exacerbation or if they were treated for a pulmonary exacerbation. Pulmonary exacerbation was defined as initiation of treatment with intravenous or oral antibiotics for 7 or more days based on respiratory symptoms at the discretion of the treating physician [24,25]. Additionally, at each visit, subjects answered questions from a checklist to ensure that all exacerbation events were captured. BODY.STATISTICAL ANALYSIS: Summary statistics were calculated for sample characteristics of each treatment group. Fisher’s exact method and Wilcoxon rank sum test was used to detect treatment group differences in baseline binary and quantitative variables respectively. Kaplan-Meier product limit method was used to estimate cumulative probability curve for time to first exacerbation in each treatment group and log rank test was used to detect group difference in the curve. Rate of exacerbation defined as number of exacerbations per person year was calculated by treatment group and negative binomial model was used to examine treatment group differences. Linear model with repeated measures were used to examine treatment group difference in FEV1, FVC, CFQ-R and GSAS over time. For participants who were withdrawn after randomization, longitudinal analyses compared each value at the start of the treatment period to the last observed value carried forward for each variable examined. BODY.RESULTS: Twenty one subjects were screened; two subjects withdrew consent before randomization, one subject was ineligible based on daily symptoms of GER (an indication for acid suppressor therapy) and one subject was ineligible due to frequency of exacerbations being above the threshold for enrollment. Of the 17 subjects who were randomized, four were unable to tolerate insertion of the pH probe but remained in the study. Fifteen subjects completed the study; all randomized subjects are included in the analysis (Figure 1). There were no significant differences between subjects randomized to placebo and those randomized to esomeprazole, though the placebo group tended toward lower lung function, more frequent exacerbations and lower body mass index (BMI) (Table 1). Of the subjects who underwent 24 hour pH probe monitoring, five of eight subjects (62.5%) in the esomeprazole group and three of five subjects (60%) in the placebo group had probe evidence of GER. There were no significant differences in baseline characteristics between subjects with and without evidence of distal GER (Table 2). Figure 1Flow diagram for screened and enrolled subjects. Table 1Baseline characteristics of subjects by treatment assignment  Esomeprazole (n = 9)Placebo (n = 8)p valueReflux present on pH probe 5/8 (62%) 3/5 (60%) 0.42 Male (%) 67 75 0.38 Pseudomonas present (%) 89 62   MRSA present(%) 0 25     Mean + SD Mean + SD   Age (years) 35.72 + 9.6 32.81 + 5.84 0.41 BMI 24.25 + 4.72 21.84 + 3.02 0.21 # exacerbations past 2 years 4 + 0 (0) 5.5 + 1.4 (SD)   FEV1 (%) 58 + 19 46 + 21 0.14 FVC (%) 74 + 20 71 + 16 0.88 FEV1/FVC 0.63 + 0.10 0.56 + 0.15 0.26 GSAS distress score 0.99 + 0.61 0.88 + 1.03 0.28 CFR-QOL score 72.28 + 10.32 77.85 + 18.86 0.34 Table 2Comparison of subjects with and without gastroesophageal reflux as measured by 24 hour ambulatory pH probe  +pH probe (n = 8)-pH probe (n = 5)p valueAge 33.8 (4.37) 37 (16.5) 0.59 FEV1 (%) 51 (17) 59 (20) 0.45 BMI 23.5 (2.7) 21.8 (5/2) 0.43 GSAS 0.65 (0.29) 0.59 (0.21) 0.88 + exacerbations previous two years 5.5 (1.4) 4 (0) 0.33 Forty one percent of 17 subjects had a pulmonary exacerbation during the study. Five of nine subjects in the esomeprazole group compared with 2 of 8 subjects in the placebo group experienced exacerbations (esomeprazole vs. placebo: odds ratio = 3.455, 95% CI = (0.337, 54.294). There was no significant difference in time to first pulmonary exacerbation between the esomeprazole and placebo groups (log rank test p = 0.3169) (Figure 2). Similarly, there was no significant difference between groups in exacerbation rate during the study period (2.04 exacerbations per person year in esomeprazole group 95% CI (1.33, 4.14) compared with 0.59 exacerbations per person year in placebo group (95% CI (0.19, 1.82), p = 0.07. There was no significant change in FEV1 percent predicted or FVC percent predicted in either group over the study period, p = 0.23 and 0.58, respectively, and there was no difference between groups in change in FEV1 or FVC percent predicted from baseline to end of study (Figure 3). GSAS and CFQ-R score did not change significantly over the study period (p = 0.27 and 0.32, respectively) and there was no difference in change in scores between the two treatment groups. Figure 2Time to first exacerbation in treatment group assigned to esomeprazole versus placebo. Log rank test p = 0.3169. Figure 3A. Forced Expiratory Volume in 1 second (FEV1) over treatment period. B. Forced Vital Capacity (FVC) over treatment period. C. Gastroesophageal Symptom Assessment Score (GSAS) over treatment period. D. Cystic Fibrosis Quality of Life – revised (CFQ-R) score over treatment period. Blue lines: esomeprazole group; mean with standard deviation. Red lines: placebo group; mean with standard deviation. BODY.DISCUSSION: Individuals with CF have many predisposing factors to the development of GER including airway hyperinflation, frequent cough, hyperalimentation, delayed gastric emptying, high fat diet and positional changes related to performance of chest physiotherapy [25]. Twenty-four hour esophageal pH monitoring has the highest sensitivity and specificity for the detection of GER and is widely regarded as the gold standard for quantifying esophageal pH. We demonstrate that the majority of patients with cystic fibrosis in our cohort have evidence of distal esophageal reflux as measured by esophageal pH monitoring despite absence of symptoms. In the small prospective study reported here, suppression of gastric acid with esomeprazole did not lead to significant improvement in pulmonary outcomes. An unanticipated finding of this study was a trend to earlier exacerbation and more frequent exacerbations in patients randomized to esomeprazole compared with placebo. In 2010, the Cystic Fibrosis Foundation Patient Registry reported that 50.7% of children less < 18 years and 48.2% of adults > 18 years were being treated with proton pump inhibitors. Though studies have suggested that treatment of GER is associated with improvement in other lung diseases, prospective studies have not been conducted in CF to determine whether reducing gastric pH has a beneficial effect on pulmonary exacerbations or other health related outcomes. The possible mechanisms whereby gastroesophageal reflux leads to respiratory symptoms in CF and other chronic lung diseases have not been established. Some investigators speculate that reflux into the esophagus, particularly in the supine position, results in intermittent aspiration of acidic stomach contents into the airways compounding the effects of the vicious cycle of inflammation, infection and progression of lung disease that has been well described in CF. Mendez, et al demonstrated that even after lung transplantation, 90% of patients with CF had evidence of GER compared with only 54% of patients who underwent lung transplant for other diseases. The majority of CF patients had evidence of proximal and distal GER [26]. Tracheal acidification has in fact been demonstrated in adults with CF while in the supine position [27]. It is further hypothesized that afferent receptors within the esophageal mucosa, when stimulated by exposure to acid, trigger outputs along motor neurons to the respiratory muscles and tracheobronchial tree, resulting in cough, bronchospasm and perhaps even increase in neutrophilic airway inflammation [28,29]. A relationship between GER and the development of obliterative bronchiolitis after lung transplantation, with improved allograft function after Nissen fundoplication has been reported by Davis and colleagues [30]. However, a large prospective study of the effect of PPIs on asthma exacerbations did not show an improvement in asthma outcomes [11]. PPIs address only the acid component of reflux, and there is evidence that non-acid reflux, such as bile salts from the small intestine, may also be lung irritants. Tamhankar and others have demonstrated that omeprazole does not reduce the number of reflux episodes or their duration, but acts to convert acid reflux to less acid reflux [31]. Doumit et al showed that among children with CF, 63% of reflux episodes were acid compared with 37% which were non acid [32]. In a study by Pauwels, et al, 56% of patients with CF had bile acids in the sputum, providing evidence for the aspiration of duodenogastric contents [25]. Furthermore, concentration of bile acids correlated with neutrophil elastase in sputum, degree of lung function impairment and need for IV antibiotic treatment. PPIs have the potential to increase the incidence of hospital and community acquired pneumonia, as demonstrated by several retrospective studies of PPI use in both the in-patient and outpatient setting [15,16]. Individuals with CF have chronic airway infections with a host of pathogens, notably Pseudomonas aeruginosa and Staphylococcus aureus. Despite widespread use of PPIs in this patient population, their safety and effect on pulmonary outcomes have not been studied. Our randomized placebo controlled double blind study of the effect of proton pump inhibitors on pulmonary exacerbations in a group of patients with CF and a known history of recurrent exacerbations was designed as a feasibility study and was underpowered to demonstrate a significant effect on respiratory outcomes. We demonstrated that in a population of patients with CF and recurrent pulmonary exacerbations, 60% of patients have asymptomatic acid GER. These results are consistent with those reported by Brodzicki et al where 55% of children with CF had GER, despite the absence of symptoms in many of those patients [33]. There was a trend toward shorter time to first pulmonary exacerbation and higher exacerbation rate in patients randomized to esomeprazole compared with placebo, despite that fact that the placebo group had more frequent exacerbations during the two years prior to study enrollment . Though the study enrolled only subjects with frequent pulmonary exacerbations (between 2 and 4 per year), there was a relatively low incidence of pulmonary exacerbations during the treatment period in that only 42% of subjects experienced an exacerbation over a thirty-six week period. This may be related to the introduction of new therapies during the study period, such as hypertonic saline and inhaled aztreonam lysine. [34,35]. Our study hypothesized that gastric acid suppression would prolong time to first pulmonary exacerbation, thus adequate gastric acid suppression was an essential component of the study design. Esomeprazole was selected because of its high potency for gastric acid suppression; the twice-daily dose of 40 mg has been shown to effectively suppress gastric acid in about 95% of patients [30]. 36 weeks was chosen for study duration to allow long enough follow up time for development of respiratory exacerbation in the majority of patients. Our study findings are limited by small sample size without adequate power to detect significant differences between subjects treated with esomeprazole compared with placebo. However, trends regarding frequency of exacerbation and time to exacerbation were consistent in the esomeprazole group. The fact that our results align with reports from several retrospective studies demonstrating an increased risk of lower respiratory tract infections in patients taking PPIs, and that patients with cystic fibrosis chronically harbor bacterial pathogens and develop recurrent pulmonary exacerbations, suggests that further investigation into the possible effects of PPIs on pulmonary infections in CF is warranted. This work was previously presented in Abstract form at the North American Cystic Fibrosis Conference 2012 [33]. BODY.CONCLUSION: Asymptomatic gastroesophageal reflux is present in the majority of patients with cystic fibrosis. Risk and benefits of acid suppressive agents in cystic fibrosis require further study. BODY.COMPETING INTERESTS: BODY.AUTHORS’ CONTRIBUTIONS: ED and PW developed the study protocol and oversaw all aspects of the study. CK, MB, ELS and NR conducted study visits and assisted with data analysis. DL and XL performed statistical analysis for the study. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2466/14/21/prepub

TITLE: Efficacy and safety of adalimumab in patients with non-radiographic axial spondyloarthritis: results of a randomised placebo-controlled trial (ABILITY-1) ABSTRACT.PURPOSE: To evaluate the efficacy and safety of adalimumab in patients with non-radiographic axial spondyloarthritis (nr-axSpA). ABSTRACT.METHODS: Patients fulfilled Assessment of Spondyloarthritis international Society (ASAS) criteria for axial spondyloarthritis, had a Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score of ≥ 4, total back pain score of ≥ 4 (10 cm visual analogue scale) and inadequate response, intolerance or contraindication to non-steroidal anti-inflammatory drugs (NSAIDs); patients fulfilling modified New York criteria for ankylosing spondylitis were excluded. Patients were randomised to adalimumab (N=91) or placebo (N=94). The primary endpoint was the percentage of patients achieving ASAS40 at week 12. Efficacy assessments included BASDAI and Ankylosing Spondylitis Disease Activity Score (ASDAS). MRI was performed at baseline and week 12 and scored using the Spondyloarthritis Research Consortium of Canada (SPARCC) index. ABSTRACT.RESULTS: Significantly more patients in the adalimumab group achieved ASAS40 at week 12 compared with patients in the placebo group (36% vs 15%, p<0.001). Significant clinical improvements based on other ASAS responses, ASDAS and BASDAI were also detected at week 12 with adalimumab treatment, as were improvements in quality of life measures. Inflammation in the spine and sacroiliac joints on MRI significantly decreased after 12 weeks of adalimumab treatment. Shorter disease duration, younger age, elevated baseline C-reactive protein or higher SPARCC MRI sacroiliac joint scores were associated with better week 12 responses to adalimumab. The safety profile was consistent with what is known for adalimumab in ankylosing spondylitis and other diseases. ABSTRACT.CONCLUSIONS: In patients with nr-axSpA, adalimumab treatment resulted in effective control of disease activity, decreased inflammation and improved quality of life compared with placebo. Results from ABILITY-1 suggest that adalimumab has a positive benefit–risk profile in active nr-axSpA patients with inadequate response to NSAIDs. BODY.INTRODUCTION: Axial spondyloarthritis may be split into two categories—ankylosing spondylitis (AS) and non-radiographic axial spondyloarthritis (nr-axSpA)—by the 1984 modified New York criteria which require the presence of sacroiliitis on plain x-rays for the classification of AS.1 2 With the availability of MRI, the presence of inflammation in the axial skeleton in the absence of radiographic changes can now be assessed.2–4 The Assessment of Spondyloarthritis international Society (ASAS) has validated new classification criteria for patients with axial SpA5 6 to establish standards that apply to patients with or without radiographic sacroiliitis by including both plain x-rays and MRI as imaging modalities.5 Although these criteria identify both AS and nr-axSpA patients, they also allow the conduct of clinical trials for the treatment of nr-axSpA which is considered an unmet medical need.5 Registry and clinical trial data indicate that patients with AS and those with nr-axSpA have comparable clinical manifestations and burden of disease, requiring treatment irrespective of the presence of radiographic damage.7–9 Non-steroidal anti-inflammatory drugs (NSAIDs) are considered first-line therapy for all patients with axial SpA.10 11 Traditional disease-modifying antirheumatic drugs (DMARDs) such as methotrexate and sulfasalazine are not effective for the treatment of axial SpA.11–14 Anti-tumour necrosis factor (TNF) agents are approved therapies for patients with AS who continue to have active disease despite NSAIDs. However, for patients with nr-axSpA there is currently no alternative treatment to NSAIDs. New treatment recommendations for patients with axial SpA, including nr-axSpA, identify anti-TNF agents as options in patients who fail NSAIDs;11 however, large phase III trials for this population have been lacking. Previous clinical trials conducted prior to the availability of the ASAS axial SpA criteria employed variable definitions of nr-axSpA and were limited by small sample sizes; nonetheless, these studies provided data showing that anti-TNF therapy is also effective in nr-axSpA.9 15 The present study was developed to further evaluate the efficacy and safety of adalimumab in the treatment of patients with nr-axSpA who have active disease despite treatment with NSAIDs. This represents the first randomised controlled clinical trial to use the ASAS axial SpA criteria in classifying patients with nr-axSpA. BODY.METHODS: BODY.PATIENTS: Eligible patients were ≥18 years of age and fulfilled ASAS classification criteria for axial SpA6 without meeting modified New York criteria for AS.1 Patients must have had active disease, exhibited by a total back pain score of ≥4 on a 0–10 cm visual analogue scale (VAS) (≥40 on a 0–100 mm VAS) and a Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score of ≥4. They must also have responded inadequately or been intolerant to one or more NSAIDs, or had a contraindication to NSAIDs based on the clinical judgment of the investigator. Patients with previous or current diagnoses of psoriasis or psoriatic arthritis or a history of inflammatory arthritis of a different aetiology were excluded. Previous exposure to biological agents was not permitted. BODY.STUDY DESIGN: ABILITY-1 (NCT00939003) was initiated in August 2009 and is an ongoing phase III randomised placebo-controlled double-blind trial conducted at 37 centres in Australia, Belgium, Canada, Czech Republic, France, Germany, Spain, The Netherlands, the UK and the USA. It was conducted in accordance with International Conference on Harmonization good clinical practices and the Declaration of Helsinki. Approval of an institutional ethics review board and voluntary written informed patient consent were obtained prior to study procedures. Eligible patients were centrally randomised using an interactive voice response system 1:1 to receive subcutaneous injections of adalimumab (40 mg every other week) or matching placebo for 12 weeks during the double-blind period. Efficacy and safety were assessed at weeks 2, 4, 8 and 12. Patients who completed the double-blind period were eligible to receive open-label adalimumab for up to an additional 144 weeks. Patients could enter the study on concomitant NSAIDs, prednisone (≤10 mg per day), methotrexate (MTX, ≤25 mg per week), sulfasalazine (SSZ, ≤3 g per day) and/or hydroxychloroquine (≤400 mg per day) or azathioprine (≤150 mg per day, but not concomitant with any other DMARD) if the doses met pre-specified stability requirements prior to randomisation and remained stable during the first 24 weeks except as medically required due to an adverse event (AE). BODY.EFFICACY ENDPOINTS: BODY.PRIMARY EFFICACY ENDPOINT: The primary efficacy endpoint was the proportion of patients who achieved an ASAS40 response at week 12. An ASAS40 response was defined as a ≥40% improvement and an absolute improvement from baseline of ≥2 units (range 0–10) in ≥3 of the following four domains: Patient Global Assessment of Disease Activity (0–10 cm VAS), pain (total back pain, 0–10 cm VAS), function (Bath Ankylosing Spondylitis Functional Index (BASFI), 0–10 cm VAS16) and inflammation/morning stiffness (mean score of items 5 and 6 of the BASDAI (0–10 cm VAS)) without any worsening in the remaining domain.17 BODY.SECONDARY EFFICACY ENDPOINTS: Secondary efficacy variables analysed at week 12 included: ASAS20,18 ASAS partial remission (ASAS PR),18 ASAS5/6,17 BASDAI (0–10 cm VAS), BASDAI50, Ankylosing Spondylitis Disease Activity Score (ASDAS),19 20 ASDAS clinically important improvement (ASDAS CII, decrease from baseline ≥1.1), ASDAS major improvement (ASDAS MI, decrease from baseline ≥2.0), ASDAS inactive disease (ASDAS ID, score <1.3),21 Maastricht Ankylosing Spondylitis Enthesitis Score (MASES, 0–13),22 linear Bath Ankylosing Spondylitis Metrology index (BASMIlin, 0–10),23 36-Item Short Form V.2 Health Survey (SF-36), Health Assessment Questionnaire modified for Spondyloarthropathies (HAQ-S)24 and Spondyloarthritis Research Consortium of Canada (SPARCC) MRI scores for the sacroiliac (SI) joints (0–72)25 and the spine (0–108).26 MRI films were scored by two independent central readers who were blinded to time point and sequence. Average scores of the readers were used. BODY.SAFETY ASSESSMENTS: Treatment-emergent AEs were defined as AEs that began or worsened after the first dose of study medication through 70 days after the last dose. BODY.STATISTICAL ANALYSIS: Efficacy variables were analysed for all randomised patients who received at least one dose of blinded study medication, but excluding seven patients from one site due to investigator non-compliance. The safety population consisted of all patients who received at least one dose of study medication. A target sample size of 194 patients (97 placebo and 97 adalimumab) was calculated to provide approximately 90% statistical power to detect a 20% difference in ASAS40 response rates between the treatment groups, based on a two-sided χ2 test with a significance level of 0.05. For categorical variables, patients with missing data at week 12 were considered to be non-responders using non-responder imputation (NRI). Last observation carried forward imputed values were used for continuous variables. Analysis of covariance (ANCOVA) adjusting for the baseline score was used to compare change from baseline at week 12 between adalimumab and placebo treatment groups. VAS data were collected on 0–100 mm scales and reported as 0–10 cm data for consistency. To evaluate the impact of baseline demographics and disease conditions on the primary efficacy endpoint, ASAS40 response at week 12 was summarised by subgroups of sex (male, female), race (white, non-white), age (<40, ≥40 years), weight (<70, ≥70 kg), symptom duration (<5, ≥5 years), baseline C-reactive protein (CRP) (normal, elevated), concomitant baseline NSAID use (yes, no) or DMARD use (yes, no), history of inflammatory bowel disease (yes, no) or uveitis (yes, no), baseline HLA-B27 status (positive, negative), past or current MRI evidence of inflammation of the SI joints according to the local radiologist/rheumatologist (positive, negative) and baseline SPARCC SI joint score (<2, ≥2). For subgroup analyses, a logistic model was used to assess treatment and subgroup interaction, with a significant interaction defined as p≤0.10. AEs were summarised as the number and percentage of patients experiencing AEs using Medical Dictionary for Regulatory Activities (MedDRA, V.13.1) system organ classes and preferred terms. BODY.RESULTS: BODY.PATIENTS: There were 192 patients randomised into the study. Due to investigator non-compliance identified at a single site, seven patients (three placebo, four adalimumab) were excluded from all efficacy analyses but were included in safety analyses. Of the remaining 185 patients, 94 received placebo and 91 received adalimumab (figure 1). Through the 12-week double-blind period, one patient from each treatment group discontinued due to AEs. One placebo patient and three adalimumab patients discontinued for other reasons, which included pregnancy, lack of efficacy or violation of entry criteria. Figure 1ABILITY-1 patient disposition. a ‘Other’ reasons for discontinuation included lack of efficacy, pregnancy or violation of entry criteria. One subject in the adalimumab group had a week 12 visit but discontinued at that visit due to a positive pregnancy test and did not receive study drug at the week 12 visit. Patient demographics were comparable between the treatment groups (table 1), with no significant differences noted between adalimumab and placebo patients. Over half of the patients (55%) were women and the mean age across both groups was 38 years. Despite a mean 10-year history of symptoms, the average time since diagnosis was only approximately 3 years prior to study entry. Table 1Baseline demographics and disease characteristics for the full analysis set and subgroups of patients fulfilling the imaging or clinical arms of the Assessment of Spondyloarthritis international Society axial SpA criteria  AllImaging (MRI+)*Clinical (HLA-B27+, MRI−)†Placebo N=94Adalimumab N=91Placebo N=43Adalimumab N=46Placebo N=48Adalimumab N=45Demographics  Female, n (%) 54 (57) 47 (52) 22 (51) 26 (57) 29 (60) 21 (47)  White, n (%) 91 (97) 91 (100) 41 (95) 46 (100) 47 (98) 45 (100)  Age (years) 38.4 (10.4) 37.6 (11.3) 38.9 (8.7) 36.8 (9.9) 38.1 (11.7) 38.5 (12.6) Disease characteristics  Symptom duration (years) 10.1 (8.8) 10.1 (9.0) 10.1 (7.2) 9.3 (9.0) 10.2 (10.1) 11.0 (9.1)  Duration since diagnosis (years) 3.0 (3.8) 2.7 (4.2) 3.2 (4.4) 2.4 (3.9) 3.0 (3.4) 3.1 (4.6)  Concomitant DMARD use, n (%) 16 (17) 17 (19) 6 (14) 5 (11) 9 (19) 12 (27)  Concomitant NSAID use, n (%) 74 (79) 72 (79) 32 (74) 38 (83) 40 (83) 34 (76) History of SpA features, n (%)  Inflammatory back pain 91 (97) 88 (97) 41 (95) 43 (93) 47 (98) 45 (100)  HLA-B27 positive 70 (74) 75 (82) 22 (51) 30 (65) 48 (100) 45 (100)  Good prior response to NSAIDs 70 (74) 64 (70) 34 (79) 32 (70) 34 (71) 32 (71)  Arthritis (past or present) 49 (52) 32 (35) 22 (51) 16 (35) 24 (50) 16 (36)  Elevated CRP‡ 36 (38) 36 (40) 15 (35) 21 (46) 19 (40) 15 (33)  Heel enthesitis (past or present) 38 (40) 36 (40) 19 (44) 17 (37) 19 (40) 19 (42)  Family history of SpA 23 (25) 28 (31) 6 (14) 9 (20) 17 (36) 19 (42)  Anterior uveitis (past or present) 10 (11) 12 (13) 6 (14) 5 (11) 4 (8) 7 (16)  Dactylitis (past or present) 10 (11) 10 (11) 7 (16) 6 (13) 3 (6) 4 (9)  Inflammatory bowel disease (past or present) 6 (6) 4 (4) 3 (7) 3 (7) 3 (6) 1 (2) BASDAI (0–10) 6.5 (1.6) 6.4 (1.5) 6.4 (1.3) 6.2 (1.6) 6.6 (1.7) 6.6 (1.4) ASDAS 3.4 (0.8) 3.2 (0.8) 3.2 (0.6) 3.3 (0.9) 3.4 (0.9) 3.2 (0.7) Patient global assessment of disease activity (0–10 cm VAS) 6.8 (1.9) 6.8 (1.8) 6.7 (1.9) 6.8 (1.9) 6.8 (1.8) 6.7 (1.7) Total back pain (0–10 cm VAS) 7.0 (1.7) 6.9 (1.8) 6.9 (1.8) 7.0 (1.9) 6.9 (1.7) 6.8 (1.7) BASFI (0–10 cm VAS) 4.9 (2.3) 4.5 (1.9) 4.6 (2.3) 4.4 (2.1) 5.0 (2.3) 4.6 (1.8) Inflammation/morning stiffness§ (0–10) 6.7 (2.1) 6.5 (2.0) 6.5 (1.9) 6.3 (2.0) 6.8 (2.3) 6.6 (1.9) CRP¶ (mg/l) 7.6 (10.2) 6.8 (11.8) 6.3 (9.1) 7.4 (9.7) 8.8 (11.3) 6.2 (13.8) Elevated baseline CRP, n (%) 37 (39) 29 (32) 15 (35) 18 (39) 20 (42) 11 (24) MASES (0–13) 3.9 (3.4) 3.5 (3.6) 3.6 (3.4) 4.0 (3.8) 4.2 (3.5) 2.8 (3.5) BASMIlin (0–10) 2.7 (1.2) 2.7 (1.3) 2.6 (1.1) 2.6 (1.3) 2.7 (1.1) 2.8 (1.2) SF-36 PCS (0–100) 33.1 (8.3) 33.9 (7.7) 32.9 (7.9) 34.0 (7.5) 33.3 (8.6) 33.8 (8.0) HAQ-S (0–3) 1.1 (0.6) 1.0 (0.6) 1.1 (0.6) 1.0 (0.6) 1.1 (0.5) 1.0 (0.5) SPARCC MRI sacroiliac joint score (0–72) 4.7 (9.9) 5.1 (9.5) 7.3 (12.9) 8.5 (12.1) 2.5 (5.7) 1.6 (3.5) SPARCC MRI spinal score (0–108) 4.6 (6.3) 4.1 (5.3) 4.4 (6.5) 4.5 (6.3) 4.6 (6.2) 3.6 (3.9) All values are mean (SD) unless otherwise indicated. N: ASDAS, 91/87 placebo/adalimumab; BASFI, 90 adalimumab; BASMI, 90 adalimumab; SPARCC SI, 91 placebo, SPARCC spine, 90 placebo. *Past or present sacroiliitis on MRI suggestive of axial SpA6 based on assessment by the local radiologist/rheumatologist. †Patients who did not have past or present sacroiliitis on MRI based on assessment by the local radiologist/rheumatologist but were HLA-B27 positive; after randomisation, three patients were found to have neither a positive MRI nor a positive HLA-B27. ‡Based on data obtained in the medical history for the presence of CRP concentration above upper normal limit in the presence of back pain. §Mean of BASDAI questions 5 and 6. ¶Combined CRP levels including both standard CRP that has been converted to mg/l units and high-sensitivity CRP values. ASDAS, Ankylosing Spondylitis Disease Activity Score; BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional Index; BASMIlin, Bath Ankylosing Spondylitis Metrology Index-linear; CRP, C-reactive protein; DMARD, disease-modifying anti-rheumatic drug; MASES, Maastricht Ankylosing Spondylitis Enthesitis Score; MRI, magnetic resonance imaging; NSAIDs, nonsteroidal anti-inflammatory drugs; SpA, spondyloarthritis; SPARCC, Spondyloarthritis Research Consortium of Canada; VAS, visual analogue scale. The most common SpA features included as components of the ASAS axial SpA criteria noted in the study population were inflammatory back pain, HLA-B27 positivity and a good prior response to NSAIDs. Despite the absence of radiographic sacroiliitis, patients with nr-axSpA had high levels of disease activity as measured by the BASDAI and ASDAS. Furthermore, there were moderate levels of functional impairment and reduction in quality of life despite low baseline BASMIlin scores, indicating minimal impairment of spinal mobility. The low mean MASES score at baseline also indicated that there was limited involvement or inflammation of entheses in the overall study population, although 72% (133/185) of patients had MASES>0 at baseline (74%, 70/94 placebo; 69%, 63/91 adalimumab). With the exception of HLA-B27 status, there were no striking differences between patients who fulfilled the imaging arm (positive MRI sacroiliitis according to the local radiologist/rheumatologist) and those who fulfilled the clinical arm (HLA-B27 positive/MRI negative) of the ASAS axial SpA criteria (table 1). However, more patients who fulfilled the clinical arm of the ASAS criteria had a family history of SpA. The SPARCC MRI scores for SI joints were also lower in patients who fulfilled the clinical arm, but the scores for the spine were comparable across groups. BODY.EFFICACY: A significantly greater percentage of nr-axSpA patients treated with adalimumab achieved the primary endpoint of ASAS40 response at week 12 (33/91, 36%) compared with patients treated with placebo (14/94, 15%; p<0.001, NRI; figure 2A). Based on subgroup interaction analyses, symptom duration, age and baseline CRP status showed significant interactions with treatment on ASAS40 response (p=0.02, p=0.05 and p=0.03, respectively; NRI). There was a greater treatment effect with adalimumab among patients with symptom duration <5 years (figure 2B), those whose age was <40 years (figure 2C) and patients who had elevated CRP levels at baseline (figure 2D). HLA-B27 status did not demonstrate a significant treatment interaction with adalimumab (p = 0.42; NRI; figure 2E). Patients achieved similar ASAS40 responses with adalimumab regardless of whether or not they had past or present sacroiliitis on MRI according to the assessment of the local radiologist/rheumatologist (p=0.65; NRI). Sensitivity analyses to further explore the influence of MRI inflammation in the SI joints were conducted using centrally-read SPARCC scores. Stratifying patients using the operational classification of positive or negative SI joint MRI inflammation based on SPARCC scores ≥2 or <2 at baseline showed a numerically greater treatment effect of adalimumab in patients with SI joint scores ≥2, but the interaction was not statistically significant (p=0.31; figure 2F). However, a significant interaction with treatment was observed based on logistic regression between continuous SPARCC SI joint scores at baseline and ASAS40 response at week 12 (p=0.046, NRI; supplementary online Figure S1). Finally, patients with either a positive MRI (SPARCC score ≥2 for either the SI joints or spine) or an elevated CRP at baseline demonstrated a greater response to adalimumab compared with placebo (41% (28/69) adalimumab vs 14% (10/73) placebo), in contrast to patients who had negative MRI of the SI joints and spine and a normal CRP at baseline (23% (5/22) adalimumab vs 20% (4/20) placebo), although the interaction was not statistically significant (p=0.13, NRI). Sex, race, weight, concomitant NSAID or DMARD use and history of inflammatory bowel disease or uveitis did not significantly affect the response to adalimumab treatment. Figure 2Percentage of patients achieving Assessment of Spondyloarthritis international Society 40 response at week 12. (A) Full analysis set; *p<0.001 for comparison of treatment response between adalimumab versus placebo. (B) Patients with symptom duration <5 years or ≥5 years. (C) Patients with age <40 years or ≥40 years. (D) Patients with or without elevated C-reactive protein (CRP) at baseline. (E) Patients with presence or absence of HLA-B27. (E) Patients with SPARCC SI joint score <2 or ≥2 at baseline. Non-responder imputation. Adalimumab treatment resulted in significantly higher percentages of nr-axSpA patients achieving clinical improvements as measured by ASAS, ASDAS and BASDAI response criteria (figure 3A). Significantly higher percentages of adalimumab-treated patients also achieved states of disease remission (figure 3B), with approximately one in four patients achieving ASDAS ID after 12 weeks of treatment with adalimumab. Figure 3(A) Percentage of patients with clinical response. (B) Percentage of patients with disease remission. Non-responder imputation. *p≤0.001 for comparison of treatment response between adalimumab versus placebo except Assessment of Spondyloarthritis international Society 20 (ASAS20) (p=0.004) and ASAS PR (p=0.01). ASDAS CII, clinically important improvement; ASDAS MI, major improvement; ASAS PR, partial remission; ASDAS ID, inactive disease. Adalimumab treatment was associated with statistically significant improvements in disease activity of nr-axSpA patients whether measured by composite indices (ie, BASDAI, ASDAS) or by individual objective measures of inflammation (ie, CRP, MRI scores) when compared with placebo (table 2). In particular, there were significant decreases in each of the individual components of the ASAS40 response criteria except for BASFI. Although there was a numerically greater improvement in BASFI with adalimumab than with placebo, the difference was not statistically significant. Based on the BASFI component of the ASAS partial remission criteria, 33% of patients (25/75) had BASFI <2 at week 12 in the adalimumab group compared with 11% (9/79) in the placebo group (p=0.001) among patients with BASFI ≥2 at baseline. Minor decreases in the BASMIlin and the MASES score were also observed but were not significantly different between treatment groups. Significant improvements in physical function and quality of life were noted with adalimumab, as determined by the HAQ-S and SF36 physical component summary scores. Table 2Clinical, functional and imaging endpoints at week 12 VariablePlacebo N=94Adalimumab N=91p ValueMean change from baselineMean change from baselineBASDAI (0–10) −1.0 −1.9 0.004 ASDAS −0.3 −1.0 <0.001 Patient global assessment of disease activity (0–10 cm VAS) −0.9 −2.2 <0.001 Total back pain (0–10 cm VAS) −1.1 −2.3 <0.001 BASFI (0–10 cm VAS) −0.6 −1.1 0.053 Inflammation/morning stiffness −1.1 −2.2 <0.001 CRP (mg/l) −0.3 −4.3 <0.001 BASMIlin (0–10)* −0.1 −0.1 0.828 MASES (0–13)* −0.8 −0.6 0.962 HAQ-S Disability index (0–3)* −0.1 −0.3 0.025 SF-36 PCS (0–100)* 2.0 5.5 0.001 SPARCC MRI SI score (0–72)* −0.6 −3.2 0.003 SPARCC MRI spinal score (0–108)* −0.2 −1.8 0.001 p Value for between-group comparisons based on ANCOVA adjusted for baseline; last observation carried forward analyses unless otherwise noted. N placebo/adalimumab: BASFI, 94/90; ASDAS, 91/87. *Observed analyses (N placebo/adalimumab): BASMIlin (93/90); MASES (93/91); HAQ-S (90/88); SF-36 PCS (93/91); SPARCC MRI SI (84/84); SPARCC MRI spine (83/85). ASDAS, Ankylosing Spondylitis Disease Activity Score; BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional Index; BASMIlin, linear Bath Ankylosing Spondylitis Metrology Index; CRP, C-reactive protein; HAQ-S, Health Assessment Questionnaire modified for Spondyloarthropathies; MASES, Maastricht Ankylosing Spondylitis Enthesitis Score; MRI, magnetic resonance imaging; SF-36 PCS, Short Form-36, V.2, Health Status Survey, Physical Component Summary Score; SI, sacroiliac; SPARCC, Spondyloarthritis Research Consortium of Canada. BODY.SAFETY: During the double-blind period, similar proportions of patients in the two treatment groups experienced any AE or an infectious AE (table 3). The most common events were nausea (8.2%), diarrhoea (7.2%) and upper respiratory tract infection (4.1%) among patients in the placebo group and nasopharyngitis (11.6%), nausea (7.4%) and headache (6.3%) in the adalimumab group. There were few serious AEs. A patient in the placebo group had a serious AE with symptoms of nausea, vomiting, chills, fever and dizziness. Three serious AEs were reported in the adalimumab group: breast dysplasia, induced abortion and acute hepatitis. The acute hepatitis was reported by the investigator as probably due to isoniazid for tuberculosis prophylaxis as the baseline visit laboratory tests prior to the first dose of study drug but after starting isoniazid showed elevated liver function tests. The case of acute hepatitis was one of four hepatic-related AEs in the adalimumab group; all other hepatic-related AEs were isolated liver function test abnormalities that were not associated with a definite diagnosis or liver pathology. There were no malignancies, opportunistic infections, tuberculosis, lupus-like syndrome, demyelinating disease or deaths through week 12. Table 3Number and percentage of patients with adverse events (AEs) during the 12-week double-blind period Placebo (N=97) n (%)Adalimumab (N=95) n (%)Any AE 57 (58.8) 55 (57.9) Serious AE 1 (1.0) 3 (3.2) AE leading to discontinuation  of study drug 1 (1.0) 2 (2.1) Infectious AE 28 (28.9) 28 (29.5) Serious infection 0 0 Malignancy 0 0 Hepatic-related AE 4 (4.1) 4 (4.2) BODY.DISCUSSION: This study is the first clinical trial to incorporate the ASAS axial SpA criteria in classifying nr-axSpA patients and the largest randomised controlled trial of an anti-TNF therapy in this population. Results from this study provide important insights into the characteristics of patients with nr-axSpA and the potential benefits of adalimumab therapy. First, the level of disease activity of nr-axSpA patients enrolled in this study was similar to those of AS patients who participated in randomised controlled trials of anti-TNF therapies. Second, significant clinical improvement was observed in nr-axSpA patients treated with adalimumab compared with placebo after just 12 weeks of therapy. Third, consistent with previous reports, the clinical response to adalimumab treatment in nr-axSpA patients was greater in patients with shorter symptom duration, younger age or presence of elevated CRP at baseline. The nr-axSpA population enrolled in this study was younger and had a greater proportion of women compared with AS populations in randomised controlled trials of anti-TNF therapies.8 27 28 However, these observations are consistent with what has been reported for other nr-axSpA cohorts, including the patient population that was evaluated to validate the ASAS axial SpA criteria.6 7 9 29 There was an average delay of 7 years between onset of symptoms to diagnosis in nr-axSpA patients in this study, which highlights the need for better ways to identify these patients. Despite the absence of radiographic sacroiliitis and the above differences in demographics compared with AS patients, patients with nr-axSpA who continued to have symptoms of active disease despite NSAIDs had comparable levels of disease activity to that of patients with AS according to BASDAI, ASDAS and total back pain.8 9 27 28 However, a smaller proportion of patients with nr-axSpA had elevated CRP at baseline compared with AS patients, and mean MRI SPARCC scores for the SI joints and spine at baseline were lower than previous reports in patients with AS.8 27 28 30 The study met its primary endpoint with 36% of patients in the adalimumab group achieving ASAS40 response at week 12 compared with 15% in the placebo group. ASAS40 is a more stringent measure of response than ASAS20 typically used in AS trials. Treatment effect with adalimumab was also significant when other composite measures were used (ie, ASAS20, ASAS 5/6, BASDAI50, ASDAS). More importantly, clinical remission was achieved by more patients in the adalimumab arm than in the placebo arm, whether defined by ASAS PR or ASDAS ID. The clinical efficacy of adalimumab was further supported by significant improvements in objective measures of inflammation (ie, CRP and the SPARCC MRI scores for both SI joints and spine). Although improvements in functionality and quality of life were noted based on the SF36 physical component summary score and HAQ-S, improvement in BASFI and BASMIlin did not meet statistical significance. This could be attributed in part to the relatively low baseline BASFI and BASMIlin scores. These results confirm previous findings from smaller trials of anti-TNF agents in nr-axSpA. In a randomised controlled trial of 46 patients with nr-axSpA, adalimumab therapy resulted in significantly better ASAS40 responses at week 12 compared with placebo.9 Likewise, in 40 patients with inflammatory back pain for 3 months to 3 years, HLA-B27 positivity and MRI evidence of sacroiliitis, patients receiving infliximab had significantly greater reduction in BASDAI and MRI scores than those on placebo at week 16.15 Subgroup analyses of the interaction between certain baseline characteristics and treatment showed that patients whose symptom duration was <5 years, age <40 or baseline CRP was elevated were more likely to achieve ASAS40 at week 12 with adalimumab. Similar observations have been reported in established AS 31–33 and were noted in a randomised controlled trial of adalimumab in 46 patients with active axial SpA without radiographically-defined sacroiliitis: patients with disease duration ≤3 years, age ≤30 or CRP>6 mg/l at baseline had a greater probability of achieving ASAS40 or BASDAI50 at week 52.9 These findings further emphasise the need to diagnose patients earlier. No other baseline variables evaluated had an impact on treatment response, including HLA-B27 status and past or present sacroiliitis on MRI according to the ASAS criteria, suggesting that adalimumab is a potential treatment option for nr-axSpA patients regardless of whether they fulfil the imaging or clinical arm of the ASAS axial SpA criteria. However, patients with active inflammation of the SI joints based on a SPARCC score ≥2 at baseline showed a numerically (although non-significant) higher response, and increasing baseline SPARCC SI joint scores were associated with a greater likelihood of clinical response. These findings suggest that the greater the extent of SI joint inflammation, the more likely a patient may benefit from adalimumab treatment. There was also a trend indicating better responses in patients with either inflammation of the SI joints or spine or an elevated CRP at baseline. Thus, objective evidence of active inflammation at baseline, such as presence of a positive MRI or an elevated CRP level, seems to be a good predictor of treatment response to adalimumab. Adalimumab was well tolerated during the double-blind period of this study. There were no notable imbalances in the occurrence of AEs between treatment groups. Safety data are consistent with what is known about the safety profile of adalimumab in AS and other immune-mediated diseases.8 34 Limitations of this study include the duration of the double-blind period, which does not allow for longer term comparison of the efficacy of adalimumab therapy with placebo in nr-axSpA patients who continue to have active disease despite NSAIDs. This study was not designed to evaluate if adalimumab therapy can prevent progression from nr-axSpA to AS. The trial was also not powered for subgroup analyses, which were further limited by uneven distribution of patients in certain subgroups (eg, HLA-B27 status). In addition, the outcome measures used in this study were validated for AS and have not been specifically developed and validated for a nr-axSpA population. As patients with nr-axSpA and those with AS are part of the same disease spectrum of axial SpA and have similar disease manifestations, the use of previously developed outcome measures for AS in this study of nr-axSpA patients was deemed to be appropriate. In summary, 12 weeks of adalimumab therapy in patients with nr-axSpA resulted in significant clinical improvements compared with placebo, providing additional evidence that adalimumab controls inflammation with a similar safety profile across a range of spondyloarthritides. Efficacy and safety results from this study suggest that adalimumab is an appropriate treatment option for active nr-axSpA patients who fail NSAIDs, especially those with objective evidence of inflammation. Longer term data would provide information on the optimal use of adalimumab in patients with nr-axSpA. BODY.SUPPLEMENTARY MATERIAL: BODY.WEB FIGURE:

TITLE: Dexmedetomidine-Fentanyl versus Midazolam-Fentanyl in Pain Management of Distal Radius Fractures Reduction; a Randomized Clinical Trial BODY.INTRODUCTION: BODY.INTRODUCTION:: Currently, using various combinations of sedative and analgesic agents has received attention for induction of sedation and analgesia due to their synergy in controlling pain and anxiety. The present study was designed with the aim of comparing dexmedetomidine-fentanyl combination with midazolam-fentanyl in this regard. BODY.METHODS:: In this randomized clinical trial, patients diagnosed with distal radius fracture who had visited the emergency department (ED) were allocated to either the group receiving the combination of fentanyl-midazolam or the one receiving dexmedetomidine-fentanyl for procedural sedation and analgesia (PSA) and were compared regarding analgesic characteristics, time to recovery and side effects. BODY.RESULTS:: 80 patients with the mean age of 42.08 ± 12.17 (18 - 60) years were randomly allocated to 2 groups of 40 (83.80% male). The 2 groups did not have a significant difference regarding baseline characteristics as well as pain severity. Mean pain score at the time of procedure was 3.47 ± 1.37 in dexmedetomidine and 2.85 ± 1.05 in midazolam group (p = 0.025). In addition, time to recovery in dexmedetomidine and midazolam groups was 6.60 ± 1.86 minutes and 12.70 ± 1.70 minutes, respectively (p < 0.001). Out of the 9 patients who experienced treatment failure, 8 (88.90%) patients were in dexmedetomidine group and 1 (11.10%) was in midazolam group (p = 0.029). Absolute risk increase rate of treatment failure in case of using dexmedetomidine instead of midazolam was 17.50% (95%CI: 4.19 – 30.81) and number needed to harm was 6.00 (95% CI: 3.20 – 23.80). BODY.CONCLUSION:: Although the combination of dexmedetomidine-fentanyl had a shorter time to recovery compared to midazolam-fentanyl for induction of sedation and analgesia, the treatment failure rate in case of using dexmedetomidine with 1 µg/kg increased 17.5% and about 1 out of each 6 patients needed a rescue dose. BODY.INTRODUCTION:: Distal radius fracture is among the common orthopedic problems and preserving the function of the joint by reduction, anatomic correction and fixating under sedation and analgesia are the priorities of treating these patients in emergency department (ED) (1). For reducing pain at the time of reduction, various methods exist including nerve block (2), hematoma block (3), and induction of sedation and analgesia using various drug compounds available to the physicians handling these patients (4, 5). Fewer side effects on the respiratory system and hemodynamic status are among the characteristics desired by emergency physicians when selecting a drug for use in induction of sedation and analgesia. Currently, using various combinations of sedative and analgesic agents has received attention in this regard due to their synergy in controlling pain and anxiety. For this purpose, the combination of midazolam and fentanyl, as a powerful and short acting synthetic opiate and a fast acting benzodiazepine with a short half-life, respectively, has been very desirable and frequently used among emergency medicine specialists (6, 7). On the other hand, dexmedetomidine as a central alpha-2 agonist that has received the official approval of American food and drug administration (FDA) in 1999 and has sympatholytic, anti-anxiety, pain relief, and analgesic effects has been considered for induction of conscious sedation (8). Findings of various studies on comparison of the sedative effects of dexmedetomidine and midazolam is indicative of their difference regarding time of recovery and side effects on the respiratory system and hemodynamics (9, 10). Although a study by Vazquez et al. indicated that the time to recovery of midazolam was twice the time to recovery of dexmedetomidine (11), another study by Zeyneloglu et al. showed completely reversed results (12). It seems that dexmedetomidine will have a more successful performance compared to midazolam in cases that sedation is required for airway procedures such as bronchoscopy (13). Therefore, the present study was designed and performed with the aim of comparing dexmedetomidine and fentanyl combination with midazolam and fentanyl combination in induction of sedation and analgesia for reduction of distal radius fracture in the ED. BODY.METHODS:: Study design and setting In the present single blind randomized clinical trial, patients diagnosed with distal radius fracture who had visited the ED of Imam Hossein and Hafte Tir Hospitals, Tehran, Iran, were studied. The patients were allocated to either the group receiving the combination of fentanyl and midazolam (midazolam group) or the one receiving dexmedetomidine and fentanyl (dexmedetomidine group) for induction of sedation and analgesia for reducing the fracture and the characteristics of the 2 combinations were compared. This study was approved by the ethics committee of faculty of medicine, Shahid Beheshti University of Medical Sciences and was registered on the Iranian registry of clinical trials (IRCT) under the number IRCT20160401027165N1. The researchers adhered to ethical principles and confidentiality of patient data throughout the study. Informed consent was obtained from the patients for participating in the study. Participants All the patients with distal radius fractures who had presented in the working shifts of the senior resident in charge of the study (including morning and night shifts in both weekdays and holidays) and aged between 18 and 60 years were included in the study without any sex limitations and using non-probability consecutive sampling. Patients who had a history of using antihypertensive or antihistamine medications, patients with head trauma and loss of consciousness, severe chest trauma, cervical vertebra trauma with unstable fracture, mental retardation, those who could not verbally communicate, hemodynamically unstable patients, those with a history of allergic reaction to drugs, addicts and those who had a history of drug abuse, pregnant women, and those with a history of cardiac disease (cardiac block and bradycardia) were excluded from the study. Intervention After selecting the patients meeting the inclusion criteria, the participants were randomly allocated to one of the study groups. After establishment of proper peripheral vein, cardiac monitoring, pulse oximetry, blood pressure monitoring and preparing complete equipment for cardiopulmonary resuscitation on the patients’ bedside, attempts were made to induce sedation and analgesia at the level of conscious sedation. The patient and the person who was responsible for statistical analysis were blind to the type of drug used. A trained nurse was in charge of preparing the 2 drug compounds used in separate syringes looking the same. In addition, a senior emergency medicine resident was in charge of the study and data gathering under the supervision of an emergency medicine specialist. We cannot be sure of the blinding of the person gathering data to the type of treatment received, due to the difference in the method of injecting midazolam and dexmedetomidine and not performing double dummy blinding. In this study, dose of dexmedetomidine (Huspiria of USA, Behestan Phamaceutical CO, Iran) was considered 1µg/kg and was injected during 10 minutes in 100cc normal saline. In addition, midazolam (Darupakhsh Co, Iran) with dose of 0.01 mg/kg was administered via slow and titrated intravenous injection. Fentanyl (Abu Ravihan Co, Iran) was prescribed with 3 µg/kg body weight dose for both groups and was administered via slow intravenous injection. Pain severity of the patients was measured and recorded using visual analogue scale (VAS) once before administration of the drugs and once before performing the procedure (10-15 minutes after drug administration). A score of 10 was considered the worst pain score and 0 was the lowest score. At least 3 points decrease in pain severity 10-15 minutes after receiving the drug was considered as treatment success and not decreasing as much was considered treatment failure. In case the pain did not decrease in a maximum of 15 minutes after drug injection, another injection was done using fentanyl with 3 µg/kg body weight dose but these patients were not excluded from the final analysis. Data gathering To gather data, a designed checklist consisting of age, sex, pain severity before intervention and 15 minutes after receiving the drug, duration of procedure, time to recovery, and probable side effects (apnea, nausea and vomiting, hypotension, and bradycardia) was used. The person in charge of data gathering was a senior emergency medicine resident under the supervision of an emergency medicine specialist. The duration of procedure was considered from the initiation of reduction until the end of fixating the reduced bone. In addition, time to recovery was considered the time interval between the end of the procedure until complete regain of consciousness and awakening of the patient Statistical analysis For analyzing data, SPSS 21 statistical software was used. For reporting data, frequency and percentage, or mean ± standard deviation were used. Minimum required sample size for performing the study was determined as 40 patients in each group considering type 1 error of 5%, 95% power, 96% and 67% probability of failure for the 2 groups (12) and the minimum clinically significant difference of 30%. To compare the results between the 2 groups, statistical tests including t-test, chi-square, and Fisher’s exact test were applied. In addition, the rate of absolute risk increase and number needed to harm of treatment failure in case of using dexmedetomidine instead of midazolam was calculated using a medical calculator and reported. Level of significance was considered to be 5%. BODY.RESULTS:: Baseline characteristics of the patients 80 patients with the mean age of 42.08 ± 12.17 (18 - 60) years were randomly allocated to 2 groups of 40 and studied (83.80% male). Table 1 compares the age and sex distribution of the patients in the 2 groups. Mean age in dexmedetomidine group was 40.65 ± 13.25 and it was 43.52 ± 10.95 years in midazolam group (p = 0.294). All the studied patients had a pain severity equal to or greater than 6 in the beginning of reduction. Mean pain severity in dexmedetomidine and midazolam groups before reduction was 8.28 ± 1.13 and 8.18 ± 1.08, respectively (p = 0.688). Response to treatment Overall, 71 (88.80%) patients experienced 3 points decrease in pain score after receiving the drug. Out of the 9 patients who experienced treatment failure, 8 (88.90%) patients were in dexmedetomidine group and 1 (11.10%) was in the midazolam group (p = 0.029). Table 2 compares mean pain severity at the time of reduction, mean duration of reduction and mean time to recovery between the 2 groups. Mean pain score at the time of reduction was not significantly different between the 2 groups from a clinical point of view; however, dexmedetomidine group had a significantly shorter time to recovery (p < 0.001). None of the patients in either group experienced any special side effects including apnea, nausea and vomiting, or hypotension. Only 3 (7.5%) patients in dexmedetomidine group experience a short episode of bradycardia, which was resolved by slowing the infusion rate from 10 minutes to 15-20 minutes without hemodynamic impairment. Absolute risk increase rate of treatment failure in case of using dexmedetomidine instead of midazolam was 17.50% (95%CI: 4.19 – 30.81) and number needed to harm was 6.00 (95% CI: 3.20 – 23.80). Table 1Comparison of age and sex distribution of the participants in the 2 studied groups Variable Group n (%) PDexmedetomidine/fentanyl Midazolam/fentanyl Sex Male 33 (85.0) 34 (82.5) 0.762 Female 7 (15.0) 6 (17.5) Age 18-45 16 (40.0) 9 (22.5) 0.120 45-55 5 (12.5) 11 (27.5) 55 ≤ 19 (47.5) 20 (50.0) Table 2Comparing mean pain severity at the time of reduction initiation (15 minutes after sedation), mean procedure duration and mean time to recovery between the 2 studied groups Variable Groups P Dexmedetomidine Midazolam Response to treatment 32 (80.0) 39 (97.5) 0.029 Treatment failure 8 (20.0) 1 (2.5) Pain severity at the time of reduction 3.47 ± 1.37 2.85 ± 1.05 0.025 Procedure duration (minutes) 12.57 ± 1.75 12.60 ± 1.78 0.950 Time to recovery 6.60 ± 1.86 12.70 ± 1.70 < 0.001 Pain severity according to Visual analogue scale. Data were presented as mean ± standard deviation or number (%). BODY.DISCUSSION:: Based on the results of the present study, although the combination of dexmedetomidine and fentanyl had a shorter time to recovery compared to midazolam and fentanyl for induction of sedation and analgesia, the treatment failure rate in case of using dexmedetomidine with 1 µg/kg increased 17.5% and about 1 out of each 6 patients needed a rescue dose. After approval of dexmedetomidine as a sedative drug, various studies have been done regarding the effectiveness of this drug and comparing it to other sedative drugs. A comparison between this drug and propofol has indicated the similar sedative effects of both drugs despite the lower effect of dexmedetomidine on the respiratory system and hemodynamics (14, 15). Senoglu et al. in a clinical trial on 40 patients in need of sedation for non-invasive ventilation, prescribed midazolam for 1 group and dexmedetomidine for the other. Their findings indicated the equal sedative effects of both drugs; however, the group that received dexmedetomidine needed less dose adjustment in comparison to midazolam (16). Dexmedetomidine has been successfully used in providing a conscious sedation state without any respiratory distress and hemodynamic instability throughout fiberoptic bronchoscopy (17). Zeyneloglu et al. compared dexmedetomidine with midazolam/fentanyl combination in inducing sedation for extracorporeal shock wave lithotripsy (ESWL) and showed that the group receiving dexmedetomidine needed more rescue doses and also more time to recovery (12). However, the findings of the present study were in line with the mentioned one regarding more need for rescue doses; however, they do not agree regarding time to recovery. In addition, Vazquez et al. compared midazolam and dexmedetomidine for induction of sedation for endoscopy of the upper digestive system and indicated that the group that received dexmedetomidine had a significantly shorter time to recovery and higher satisfaction. In this study, no difference was reported between the 2 drugs regarding side effects and sedative properties (11). The findings of this study was in agreement with the results of the present study, yet as reported in the results section, the patients receiving dexmedetomidine had a significantly higher rate of treatment failure and more need for a rescue dose. Regarding the side effects of these drugs on respiratory system and hemodynamics there isn’t much of an agreement between the studies. Although the findings of the study by Frolich et al. indicate the superiority of midazolam for having less effect on the mentioned systems (10), Shukry et al. believe that when less effect on the airway and need for faster awakening are priorities for the in-charge physician and the patient, dexmedetomidine seems to be a proper choice (9). The reason for these differences could be the different ethnic characteristics of the studied patients, different areas in which the drugs were used, different doses of drugs, different drug compounds and…. However, there is not much experience regarding use of dexmedetomidine specially in ED and for induction of sedation and analgesia and to reach a final decision on points such as time to recovery, side effects of the drug on hemodynamics and respiratory system as well as success and failure rates, further studies are required. Performing multi-center studies with a big sample size and considering all the principles of clinical trials might help in this regard. Limitations Not using double dummy blinding and the probability of the person gathering data being aware of the drugs used can be mentioned as the most important limitations of this study. Additionally, not following patients regarding probable digestive and other side effects during the hours after sedation are among other limitations of the present study. BODY.CONCLUSION:: Based on the results of the present study, although the combination of dexmedetomidine and fentanyl had a shorter time to recovery compared to midazolam and fentanyl for induction of sedation and analgesia, the treatment failure rate in case of using dexmedetomidine with 1 µg/kg increased 17.5% and about 1 out of each 6 patients needed a rescue dose.

TITLE: Heme iron polypeptide for the treatment of iron deficiency anemia in non-dialysis chronic kidney disease patients: a randomized controlled trial ABSTRACT.BACKGROUND: Anemia secondary to iron deficiency is common in patients with non-dialysis dependent chronic kidney disease (ND-CKD) but it is unclear if oral supplementation is as effective as intravenous (IV) supplementation in re-establishing iron stores. The purpose of this study was to determine if oral Heme Iron Polypeptide (HIP) is as effective as IV iron sucrose in the treatment of iron-deficiency anemia for patients with ND-CKD. ABSTRACT.METHODS: Forty ND-CKD patients were randomized; 18 to HIP 11 mg orally 3 times per day and 22 to IV iron sucrose 200 mg monthly for 6 months. Baseline clinical and laboratory data were collected for all patients. The primary and secondary outcomes for the study were hemoglobin (Hgb) concentration and iron indices [ferritin and percentage transferrin saturation (TSAT)] at the end of 6 months respectively. Adverse events were also compared. ABSTRACT.RESULTS: The baseline demographic characteristics and laboratory values were similar for the two groups. After 6 months of treatment, Hb in the HIP group was 117 g/L and 113 g/L in the IV sucrose group (p = 0.37). The TSAT at 6 months was not different between the two groups {p = 0.82}but the serum ferritin was significantly higher in the IV iron sucrose group {85.5 ug/L in HIP and 244 ug/L; p = 0.004}. Overall adverse events were not different between the groups. ABSTRACT.CONCLUSION: HIP is similar in efficacy to IV iron sucrose in maintaining hemoglobin in ND-CKD patients with no differences in adverse events over 6 months. It is unclear if the greater ferritin values in the IV iron sucrose group are clinically significant. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov: NCT00318812 BODY.BACKGROUND: Anemia develops early during chronic kidney disease (CKD) and is associated with increased cardiovascular morbidity, mortality and decreased quality of life [1-3]. Iron deficiency is common in patients with CKD which limits the effectiveness of erythropoiesis-stimulating agents (ESA) [4-7]. The estimated prevalence of iron deficiency ranges from 25 to 70% [8,9]. Importantly iron has many other physiologic functions that may be important for overall health such as immune function, thermoregulatory performance, energy metabolism, and exercise or work performance [10]. To replete iron stores, iron can be administered either orally or intravenously (IV). Although oral iron is less expensive, easier to administer, and may be safer, IV iron enables the administration of larger doses of iron and is better tolerated by some patients [11]. The main adverse reactions to oral iron are gastrointestinal and may limit adherence and dose [12]. The most feared adverse reaction to IV iron is anaphylaxis, which is more common with iron dextran than with other preparations [13,14]. In addition, there are concerns that IV iron may accelerate kidney damage in patients with CKD not on dialysis therapy, promote infections by supplying iron to pathogenic bacteria, enhance atherosclerosis by generating oxidative stress, and cause endothelial damage [15-18]. In hemodialysis patients several different studies, including a randomized controlled trial, have consistently demonstrated that intravenous iron supplementation is superior to oral iron replacement with respect to enhancing body iron stores, augmenting hemoglobin levels and reducing ESA requirements [19-21]. Among patients with ND-CKD, by contrast, evidence for an optimal iron replacement strategy, safety, and test utility is less clear. There is controversy about when to start iron supplementation, target values for ferritin and TSAT such that the 2008 Canadian Society of Nephrology guidelines for management of iron deficiency anemia are Grade D [22]. There is also ongoing controversy as to whether iron supplementation is best administered orally or intravenously in ND-CKD and peritoneal dialysis patients [23]. There are only a few comparative studies between IV iron infusion and oral iron supplementation and the conclusions are conflicting [24-28]. Heme iron polypeptide (HIP) is a new generation oral iron which uses the heme porphyrin ring to supply iron to sites of absorption in the intestinal lumen. In comparison with the other standard iron preparations, preliminary evidence suggests that HIP may represent a promising new strategy for oral iron replacement [29-31]. For this reason, we performed a randomized controlled trial to determine if oral HIP is as effective as IV iron sucrose in the treatment of iron-deficiency anemia for patients with ND-CKD. BODY.METHODS: BODY.STUDY DESIGN: This was a single blind (investigator), randomized controlled trial performed at the Ottawa hospital from May 2007 to February 2011(NCT00318812). Randomization was via a computer generated sequence; group allocation was stored in sealed opaque sequentially numbered envelopes. The study protocol and all amendments were approved by the Ottawa Hospital Research Ethics Board. The original protocol was to include patients with an eGFR < 30 mls/minute and Hgb of 90–110 but was modified secondary to recruitment challenges. All ND-CKD patients > 18 years old with an estimated glomerular filtration rate (eGFR) ≤60 ml/min/1.73 m2 with anemia [90–120 g/L (females) 90–135 g/L (males)] and iron indices lower than the CSN recommended targets (serum ferritin < 100 ucg/L or TSAT < 20%) were invited to participate. Patients were excluded if they had received parenteral iron therapy or blood transfusion within the last 3 months, were pregnant, or had a history of recent malignancy, infection, GI bleed or major surgery. If the patient was already on an oral iron preparation, the preparation was stopped and the patient was included in the study after a wash out period of two weeks. Patients were also excluded if serum folate or vitamin B12 levels were below the normal limits (< 15 nmol/L, <133 pmol/L respectively). If the participant was being treated with an ESA, the medication was continued and the dose was adjusted by the blinded study investigator to maintain Hgb from 100-120 g/L. If the participant was not on an ESA at study entry, once the participant was iron replete (TSAT 20-50% and ferritin 100-500 ucg/L), if the Hb was <100 g/L, an ESA was started. BODY.MEASUREMENTS: Laboratory tests were done at the Ottawa Hospital. Hemoglobin was measured from plasma using spectrophotometric scan with a coefficient of variation of 4%. Ferritin was measured with an automated immunoassay with a CV of 5%. Iron and transferrin were measured with the ferrozine method with a CV of 3% and immunonephelometry with a CV of 5% respectively. BODY.TREATMENT AND FOLLOW UP: After providing informed consent, patients were randomized to receive IV iron sucrose 200 mg monthly or HIP 11 mg orally three times a day for total of 6 months. Absorption of HIP in chronic kidney disease is approximately 18.6% such that 33 mg per day is roughly equivalent to 200 mg IV iron sucrose [32]. Baseline clinical data and laboratory investigations were collected as per the protocol at the time of enrollment. Patients were followed monthly for compliance (pill counts) and possible adverse effects (standardized questionnaire) in both groups. The questionnaire specifically asked patients to quantify (none, somewhat/occasionally, a lot/often) if they experienced constipation (<1 bowel movement per 2 days), diarrhea (> 3 bowel movements per day), bloating, nausea, cramps, indigestion, muscle cramps, episodes of low blood pressure and skin rash at 2, 4 and 6 months. Hemoglobin was repeated monthly and iron indices were repeated every two months. Premature withdrawal was defined as initiation of renal replacement therapy, blood transfusion, non-adherence (refusal to take study medication) or withdrawal of consent. BODY.OUTCOME: Summary descriptive statistics were calculated to describe the study patient population using SAS enterprise (version 4.2). Results are expressed as median and interquartile range for continuous data and percentage and frequency for categorical data. The primary outcome was a comparison of Hb concentration at 6 months between the two groups using the Wilcoxon two –sample test. A similar analysis was done for the secondary outcome measures (serum ferritin, TSAT). Intent to treat analysis was utilized. Where there was missing data, the last value was carried forward for any patients with a premature withdrawal from the study protocol for whatever reason. We also examined the requirement for erythropoietin and adverse events with the medication. BODY.RESULTS: Between May 2007 to February 2011, 55 patients were consented to participate in the study and 40 patients were randomized to the treatment groups (Figure 1). There were 22 patients in the IV iron sucrose group and 18 patients in the oral HIP group. Demographics and baseline characteristics of the study population were similar in the two groups as shown in Table 1. Figure 1Trial flow. Table 1Baseline characteristics of both groups (median/interquartile range) ParameterHIPIV sucrosep-valueNumber of patients (Randomized) 18 22   Age(years) 76 (66–83) 66 (58–76) 0.10 Sex Male −13 Female −5 Male −12 Female −10 0.33 Race Caucasians −16 Africans −1 Others −1 Caucasians- 15 Africans-6 Others-1 0.12 Blood pressure (mm Hg) Systolic 130 (122–140) 131 (124–140) 0.85 Blood pressure (mm Hg) Diastolic 67 (62–75) 68 (60–72) 0.76 Hemoglobin(g/L) 110.5 (104–119) 108.5 (102–117) 0.39 Serum ferritin(ug/L) 71 (40–143) 67 (27–100) 0.59 TSAT 17 (14–20) 16.5 (10–20) 0.37 Patients on ESA* 7 6 1.0 Average ESA dose (ug/month) 60(60–80) 80(60–100) 0.20 Serum creatinine(umol/L) 246.5 (206–362) 216.5 (176–351) 0.48 Glomerular filtration rate(ml/min/1.73 m2) 20.5 (12–26) 23 (18–33) 0.39 Serum albumin 38 (35–39) 38.5 (36–41) 0.58 Intact PTH 13.05 (6.1-20.1) 13.3 (7.9-16.75) 0.93 Serum phosphate 1.28 (1.07-1.42) 1.28 (1–1.54) 0.96 Cause Of ESRD Diabetes – 6 Ischemic nephropathy-3 Hypertension – 3 Unknown – 3 Others – 3 Diabetes −9 Ischemic nephropathy-6 Hypertension – 1 Unknown – 5 Others – 1 0.22 The baseline hemoglobin was 110.5 g/L {inter quartile range (IQR): 104–119} in the HIP group and 108.5 g/L (IQR:102–117) in the IV iron sucrose group. The baseline serum ferritin was 71 ug/L (IQR: 40–143) in HIP group and 67 ug/L (IQR: 27–100) in IV iron sucrose group. Baseline serum TSAT was 17% (IQR14-20) in HIP group and 16.5% (IQR 10–20) in IV iron sucrose group. The eGFR was comparable between both groups {20.5 ml/min/1.73 m2 (IQR:12–26) in HIP and 23 ml/min/1.73 m2 (IQR:18–33) in IV iron sucrose group}. Similar numbers of patients who were already on ESA treatment. Fourteen of eighteen (78%) patients completed the study as per protocol in the HIP group and 19 of 22 (86%) in the IV iron sucrose group. After 6 months of treatment there were increases in the Hb, TSAT and serum ferritin in both the groups compared to their baseline values (Table 2). There was no difference in the Hb in the HIP group was 117 g/L and 113 g/L in the IV sucrose group at 6 months (p = 0.37; Table 3). Among iron indices, the TSAT at 6 months was also similar in both the groups {21.5%(17–29) in HIP and 21.5% (17–27) in IV sucrose; p = 0.82}, whereas serum ferritin was significantly higher in the IV iron sucrose group compared to HIP group {85.5 ug/L (44–104) in HIP and 244 ug/L (71.5-298); p = 0.004}. In the oral HIP group, in addition to the 6 patients who were on an ESA at study entry, one more patient was started on ESA by study completion. In the IV iron sucrose group, one of the 6 patients was able to discontinue ESA therapy. Table 2Change in Hgb, ferrtin and TSAT from baseline to 6 months by treatment group ParameterHIP baselineHIP 6 mp-valueIV iron sucrose baselineIV iron sucrose 6 mp-valueHgb 110.5 117 0.15 108.5 113 0.23 (104–119) (110–128.8)   (102–117) (107.5-120.3)   Ferritin 71 85.5 0.81 67 244 0.003 (40–143) (44–104)   (27–100) (71.5-298)   TSAT 17 21.5 0.05 16.5 21.5 0.04   (14–20) (17–29)   (10–20) (17–27)   Table 3Primary and secondary outcomes: hemoglobin, serum ferritin, TSAT and ESA requirement at 6 month ParameterHIPIV sucrosep-valueHgb (g/L) 117 (110–128.8) 113 (107.5-120.3) 0.37 Serum ferritin (ug/L) 85.5 (44–104) 244 (71.5-298) 0.004 TSAT (%) 21.5 (17–29) 21.5 (17–27) 0.82 Average ESA dose at 6 month (ug/month) 60 (7 patients) 50 (5 patients) 0.56 Three patients in IV iron sucrose and 4 patients in oral HIP group withdrew from the study. In the IV iron sucrose group one patient was non adherent to the study protocol, one patient required blood transfusion and one had surgery requiring withdraw from the study. In HIP group, one patient was non adherent and 3 patients discontinued secondary to new or worsening abdominal cramps. Overall adverse effects are similar between both the groups (Table 4). Four patients in each group had more than one adverse event. Gastrointestinal complaints were the most common adverse effects in both groups with constipation and abdominal cramps being the most common in HIP group and constipation in IV sucrose group. Symptomatic hypotension occurred in 3 patients in the IV iron sucrose group during infusion (13%). Table 4Adverse events ParameterHIP (18)IV sucrose (22)> 1 Adverse event 4 4 Constipation 5 4 Diarrhoea 2 3 Bloating sensation 3 2 Abd cramps 5 3 Nausea 2 2 Dyspepsia 1 3 Muscle cramps 5 2 Symptomatic hypotension 0 3 Skin rash 1 0 Overall 28 26 BODY.DISCUSSION: In our single blinded study comparing 11 mg 3 times per day oral HIP to 200 mg IV iron sucrose monthly, we did not find any difference in Hb after 6 months of therapy. The TSAT improved in both groups and was not statistically different between the patients treated with HIP or IV iron sucrose. However, the ferritin increased more in the IV iron sucrose group and this was statistically significant. Adverse events were similar in both groups. Anemia develops early during CKD and is associated with increased cardiovascular morbidity, mortality and decreased quality of life for CKD patients [1-3]. As CKD progresses, Hgb falls because of a decrease in erythropoietin production as well as iron deficiency that develops secondary to decreased absorption and increased loss. Although iron stores can be restored with either intravenous or oral iron, both therapies have potential risks and benefits. Intravenous iron may be convenient for some patients (especially those on hemodialysis) and may be associated with less gastrointestinal side effects [11,12]. However, intravenous iron is expensive and may be associated with hypotension, serum sickness type reactions and anaphylaxis [13,14]. Oral iron may be more convenient for ND-CKD and peritoneal dialysis patients, but data on the efficacy of oral iron is conflicting. HIP is produced by hydrolysis of bovine hemoglobin resulting in a highly soluble heme moiety that contains more than 1% iron. Since heme is absorbed via a different receptor than non heme (ionic) iron, the absorption kinetics and gastrointestinal side effect profiles of HIP and ionic iron are dissimilar [29,30]. Administration of HIP to 14 healthy subjects was associated with fewer side effects and significantly higher bioavailability compared with nonheme iron [31]. HIP increased serum iron levels 23 times greater than ferrous fumarate on a milligram-per- milligram basis [31]. Hallberg et al. has also shown enhanced absorption of heme iron compared to iron salts even in subjects with serum ferritin levels greater than 400 ng/mL (898 pmol/L) [33]. Although we did not compare HIP to another non-heme iron, we were able to show that supplementation of HIP to patients with ND-CKD was able to maintain Hb and improve measures of iron indices over a 6 month period. The gastrointestinal adverse events were not greater in the HIP group than the IV iron sucrose group. Our study results are consistent with a study published by Nissenson et al. on hemodialysis patients [34]. They performed an open-label, pre-test/post-test trial of HIP (1 tablet tid) administered instead of intravenous iron to 37 ESA-treated hemodialysis patients over a 6 34]. In ND-CKD anemia studies, 7 randomized controlled trials comparing the efficacy of IV iron to oral iron have been reported and yielded contradictory results [24-28,35,36]. The studies differed in several important ways including baseline Hb levels, study duration, iron status of the patients, sample size and type of IV iron preparations. In the meta-analysis by Rozen-Zvi et al., there was a small improvement in Hb concentration in patients treated with IV iron compared to oral iron [0.31 g/dl (0.09 to 0.53)], the clinical significance of this small difference is questionable [37]. In our study HIP, was compared with IV iron sucrose at doses that were considered roughly equivalent over 6 month duration. Under these conditions, HIP appeared to have similar efficacy in maintaining hemoglobin with no increase in gastrointestinal side effects. However, similar to previous randomized studies, the serum ferritin was significantly higher in IV iron group, in spite of similar TSATs in the HIP group. A similar result was seen in the recently completed HEMATOCRIT trial in which the serum ferritin was also higher in peritoneal dialysis patients treated with ferrous sulfate compared to HIP [38]. It is unclear if the increased ferritin is clinically significant. However, the ability to withdraw the ESA in one patient in the IV iron sucrose group but not in the HIP iron group requires further study. There are several limitations to our study. We had limited ability to detect a difference in Hgb values due to our small sample size (power 0.56). In designing a non-inferiority trial of HIP versus IV iron sucrose with a difference of 2 g/L in the mean of the Hb values would require 694 patients. The difficulties with recruitment and the lack of interest in participation in the study suggest that a repeat study aiming for a larger ‘N’ would not be feasible at our centre. We also did not examine the potential effects on oxidative stress between the two different types of iron and the effect of iron preparations on eGFR [14]. Since the maximum follow up has been 6 months in all the studies including our study, it limits our ability to draw conclusions regarding the long term consequences of different treatment regimens on Hb levels, progression of CKD (eGFR) and clinical outcomes, such as mortality, cardiovascular outcomes, and quality of life. Long term follow-up is especially important given the concerns about oxidative stress, infection risk, and cardiovascular morbidity and mortality secondary to the free iron released into the circulation from the IV preparation [12,15-18]. The ongoing randomized study by Agarwal et al., in which the effect of oral versus IV iron on GFR and proteinuria will be assessed over a 2 year period may address a couple of the issues [39]. BODY.CONCLUSIONS: In conclusion, we have shown in this single blinded randomized controlled trial that HIP was as effective in maintaining Hb concentration in ND-CKD patients as IV iron sucrose over a 6 month period. The results of the ongoing randomized studies with longer follow up are required to answer the important questions related to morbidity and mortality. Further studies are also required to determine the optimal time to intervene with iron therapy since iron also has other physiologic functions. BODY.COMPETING INTERESTS: None of the authors have any conflicts of interest to declare with respect to this study. The results of this study have not been published elsewhere except in abstract form. BODY.AUTHORS’ CONTRIBUTIONS: Study design and implementation – AC, JLD, DLZ. Data Analysis –. SPN, AA. Manuscript Preparation – APN, DLZ. All authors review and acceptance of final version of the manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2369/14/64/prepub

TITLE: Adverse Effects of Amoxicillin for Acute Lower Respiratory Tract Infection in Primary Care: Secondary and Subgroup Analysis of a Randomised Clinical Trial A European placebo-controlled trial of antibiotic treatment for lower respiratory tract infection (LRTI) conducted in 16 primary care practices networks recruited participants between November 2007 and April 2010, and found adverse events (AEs) occurred more often in patients prescribed amoxicillin compared to placebo. This secondary analysis explores the causal relationship and estimates specific AEs (diarrhoea, nausea, rash) due to amoxicillin treatment for LRTI, and if any subgroup is at increased risk of any or a specific AE. A total of 2061 patients were randomly assigned to amoxicillin (1038) and placebo (1023); 595 (28%) were 60 and older. A significantly higher proportion of any AEs (diarrhoea or nausea or rash) (OR = 1.31, 95% CI 1.05–1.64, number needed to harm (NNH) = 24) and of diarrhoea (OR 1.43 95% CI 1.08–1.90, NNH = 29) was reported in the amoxicillin group during the first week after randomisation. Subgroup analysis showed rash was significantly more often reported in males prescribed amoxicillin (interaction term 3.72 95% CI 1.22–11.36; OR of amoxicillin in males 2.79 (95% CI 1.08–7.22). No other subgroup at higher risk was identified. Although the study was not powered for subgroup analysis, this analysis suggests that most patients are likely to be equally harmed when prescribed antibiotics. BODY.1. INTRODUCTION: Lower respiratory tract infection (LRTI) is the most common reason for consulting a general practitioner (GP) [1,2]. LRTIs are often treated with antibiotics, even though this is not generally supported by guidelines and recommendations [2,3,4,5,6]. Many trials and observational studies have found no or little benefit of antibiotic treatment for an acute cough [7]. If an antibiotic is prescribed, amoxicillin is the recommended first-line treatment for LRTI [8]. Amoxicillin is the most commonly used broad-spectrum penicillin accounting for an average 40% of the total outpatient antibiotic use in Europe [8,9,10]. All medications have known adverse events (AEs) and antibiotics are no exceptions [11]. Although most antibiotics are generally considered safe, and most AEs are moderate to mild, some antibiotics have been associated with life-threatening AEs [12]. AEs are generally poorly reported in trials, and their true incidence is thought to be much higher than reported in trials [13]. In primary care, a shared decision consultation should include both the benefits and potential harms of the (antibiotic) treatment prescribed [14]. The European multicentre randomised placebo-controlled trial (RCT) of amoxicillin for LRTI in adults in primary care was performed by the GRACE (Genomics to combat Resistance against Antibiotics in Community-acquired LRTI in Europe; http://www.grace-lrti.org) Network of Excellence. The GRACE trial identified significantly more AEs (diarrhoea or nausea or rash) in the amoxicillin group compared to the placebo group (AEs in week one and week two after antibiotic administration) [15]. However, it was not clear whether this applies to each specific AE, or whether particular subgroups of patients suffer more AEs than others. To better inform primary care clinicians and their patients, this secondary analysis of the GRACE trial aims to provide estimates of any and each specific AE (diarrhoea, nausea, and rash) of amoxicillin, and identify subgroups of patients that are more at risk for any or a specific AE. BODY.2. RESULTS: BODY.2.1. PARTICIPANT’S DISTRIBUTION BY SUBGROUPS: A total of 2061 patients were recruited and randomly assigned to amoxicillin (1038) and to placebo (1023). Of the total, 595 (28%) were 60 years and older, 836 (40%) were males, 579 (28%) current smokers, and 470 (23%) reported symptoms of anxiety/depression on the day of consultation. Regarding the medication history, 283 (14%) had antibiotic treatment in previous 6 months, 1056 (51%) reported over-the-counter (OTC) treatment before consulting a general practitioner, 1342 (65%) were prescribed treatment other than study medication during their consultation. These other medications included 479 (23%) antihypertensive/diuretics, 201 (10%) oral bronchodilators, 171 (8%) steroids, 202 (10%) antidepressant/benzodiazepines, 174 (8%) non-steroidal anti-inflammatory drugs, and 451 (22%) influenza vaccines. BODY.2.2. SUBGROUP ANALYSIS OF ADVERSE EVENTS: For the whole cohort, at the end of week one, a significantly higher proportion of any AE (diarrhoea or nausea or rash) was reported in the amoxicillin group compared to placebo (OR = 1.31, 95% CI 1.05–1.64) (Table 1). The number needed to harm (NNH) was 24, i.e., on average, for every 24 patients receiving amoxicillin, one additional patient had reported any AE due to the antibiotic at the end of week one. Any AE was reported significantly more often in patients ever being a smoker (OR = 1.41, 95% CI 1.04–1.90), and patients using OTC treatment before the consultation (OR = 1.44, 95% CI 1.09–1.91), but the interaction terms were not statistically significant. In patients with depression/anxiety on the day of consultation (OR = 1.49, 95% CI 0.99–2.22) and in those on any medication other than study medication (OR = 1.29, 95% CI 0.98–1.69) the odds ratios were borderline significant, but the interaction terms were not significant. This indicates that compared to the whole cohort, no subgroups were at higher risk of any AE (diarrhoea or nausea or rash) due to amoxicillin. Analysing each specific AE, diarrhoea was present significantly more often among patients in the amoxicillin group, compared to those in the placebo group (OR 1.43 CI 1.08–1.90, Table 2) (NNH: 29). Diarrhoea was significantly more often reported by patients 60 years and over (OR 1.97, 95% CI 1.09–3.54), current smokers (OR 2.07, 95% CI 1.15–3.76), ever being a smoker (OR 1.79, 95% CI 1.21–2.65), on OTC treatment before their consultation (OR 1.76 95% CI 1.23–2.53), and on antihypertensive/diuretics (OR 2.27, 95% CI 1.27–4.05). However, the interaction terms were not significant. Nausea was not associated with amoxicillin treatment for either the whole cohort or any subgroup of patients (Table 3). Rash was only significantly more often reported by males (interaction term 3.72, p = 0.021; odds ratio in males 2.79 (95% CI 1.08–7.22) (Table 4). BODY.3. DISCUSSION: BODY.3.1. SUMMARY: To the authors’ knowledge, this is the first subgroup analysis of any and specific AEs reported in RCTs of antibiotics for LRTI. Diarrhoea was significantly more likely to be reported in the amoxicillin group compared to the placebo group. No specific subgroups were at higher risk of any or a specific AE due to amoxicillin, apart from males in the amoxicillin group reporting rash significantly more often. BODY.3.2. STRENGTHS AND LIMITATIONS: Our results are based on data from the largest RCT of antibiotics for acute LRTI in general practice to date [15]. Its primary objective was not identifying the incidence of AEs. RCTs, such as the GRACE trial, are not always prospectively powered for subgroup analysis of AEs [16]. Accordingly, subgroup analyses with multiple comparisons are often underpowered, with a greater risk of the false negative results (type II error). Large sample sizes are needed for robust subgroup analysis, which may only be possible by combining trial results in a meta-analysis [17]. BODY.3.3. COMPARISON WITH EXISTING LITERATURE: Reporting guidelines on RCTs indicate that more details on AEs of medication should be documented and reported to the concerned authority [18]. However, AEs occurring during a trial are often underreported, in particular, when reporting results in trial publications. Underreporting may be a result of poor monitoring, missing data, or unclear case definitions [19]. An important consequence of underreporting of AEs is a misinterpretation of the intervention’s effects, particularly its harms [20]. Although the GRACE trial captured AEs from the study medication, the reported AEs were limited to diarrhoea, nausea, and rash. A review paper identified that candidiasis was significantly associated with amoxicillin use [13], and patients treated with amoxicillin were twice as likely, compared to placebo, to report diarrhoea [13,19]. As in the previously published paper from GRACE trial [15], the current study showed significantly more AEs (diarrhoea or nausea or rash) in amoxicillin group compared to placebo. The calculation of any AEs in the previous paper covered the first two weeks after the antibiotic was prescribed, however, this paper reports on any AEs in the first week while patients were taking antibiotics. This study also showed a higher risk overall of diarrhoea in the amoxicillin group compared to the placebo group. Even though diarrhoea was more often reported in the treatment group for patients 60 years and older, smokers, patients taking OTC treatment before consulting a GP, patients on antihypertensive or non-steroidal anti-inflammatory drugs and those who received an influenza vaccine, no particular subgroup was at higher risk of AEs. The presented analysis showed that males in the amoxicillin group reported rash significantly more often compared to males in the placebo group. Skin reactions were also associated with amoxicillin use [21]. A borderline significant interaction term was observed for patients reporting anxiety/depression on the day of the consultation. These (borderline) significant results may be due to multiple testing, and with more conservative p-values, and these results would not be considered significant [22]. Sensitivity analysis did not alter our conclusions. BODY.4. MATERIALS AND METHODS: BODY.4.1. STUDY DESIGN AND PATIENTS: The GRACE trial was performed in 16 primary care research networks in 12 European countries. Details of the study design, patient inclusion, and recruitment were previously published [15,16]. In summary, the study was conducted between November 2007 and April 2010, and recruited adult patients with LRTI that were randomly allocated to receive either 1 g of amoxicillin or placebo three times a day for 7 days. BODY.4.2. DATA COLLECTION: Data was collected using (a) a case record form (CRF), (b) a symptom diary, and (c) a short version of the diary. The latter was used to collect key outcome variables and AEs during a standardised phone call after 4 weeks, if participants had not returned their diary. For this subgroup analysis, we used information on antibiotic treatment in the previous six months, any medication during the study period, history of regular use of inhaled bronchodilators, steroids, antihypertensive/diuretics, benzodiazepines/antidepressant, oral non-steroidal anti-inflammatory drugs, or influenza vaccination recorded by the responsible clinician in the CRF. The symptom diary was completed by the patient every day from day one, i.e., day of consultation and inclusion, until resolution of symptoms, up to a maximum of 28 days. This diary has previously been validated, is sensitive to change, and internally reliable [23]. Specific AEs, such as diarrhoea, nausea, and rash, were recorded at the end of week one and week two, and over-the-counter (OTC) treatment was recorded on day one. Anxiety and depression related questionnaires were completed by patients on the day of consultation (day one), and at the end of every week for four weeks. For the purpose of this study, we used anxiety and depression reported on day one. All information was collected blind to treatment allocation. All data collection forms were translated into relevant local languages and back-translated to ensure consistency. BODY.4.3. OUTCOMES IN THE STUDY: The primary outcomes include the presence of specific AEs, diarrhoea, nausea, and rash. We also created another dichotomous outcome variable for the presence of “any reported AE” for those who had reported either diarrhoea or nausea or rash at the end of week one. Amoxicillin was administered for seven days in the study, and all AEs reported during week one were included in the primary outcome. Subsequent reported AEs was excluded from the analysis. BODY.4.4. SAMPLE SIZE CALCULATION: As this is a secondary analysis of previously collected data, sample size calculations are no longer relevant. Considering the proportion of any adverse events caused by amoxicillin [15], a subgroup sample size of 136 patients allows detection of a 15% absolute difference in AEs (17.5% versus 2.5%, with 80% power, α = 0.05 (G* power Version 3.1.9.2). Similarly, 302 patients would allow the detection of a 10% absolute difference. Analyses in smaller subgroups were considered underpowered, and were only reported for comprehensive purposes. BODY.4.5. STATISTICAL ANALYSIS: The subgroup analyses of the AEs were not pre-specified. For any and each specific AE, we estimated the effect of amoxicillin using logistic regression analysis in Stata (version 13). Subgroup analyses were performed separately for any and each specific AE. The interaction between a particular subgroup (for example, males) and the intervention (in this case amoxicillin) concerns the difference in AEs (of amoxicillin) among the patients in that particular group (males), compared to patients who are not (females). The interaction term is the variable introduced into the statistical model to allow estimation of the size of that difference. The odds ratio in the subgroups estimates the difference in AEs between patients on amoxicillin and those on placebo. The specific subgroups were gender (male/female), age (60 years and older/less than 60 years), and yes/no groups for current and ever smoking, depression/anxiety on the day of the consultation, over-the-counter (OTC) treatment before consultation, antibiotics used in previous six months, use of any medication other than study medication, use of oral bronchodilators, on regular oral or inhaled steroids, on antihypertensive/diuretics, on antidepressant/benzodiazepams, on non-steroidal anti-inflammatory drugs and vaccinated against influenza. Sensitivity analysis was performed by recoding missing information on an AE as the absence of that AE. BODY.4.6. ROLE OF FUNDING SOURCE: GRACE was funded by the European Commission’s Framework Programme 6 (LSHM-CT-2005-518226). The work reported in this publication has been financially supported through TRACE (Translational Research on Antimicrobial resistance and Community-acquired infections in Europe; www.esf.org/trace). The researchers are independent of all funders. BODY.4.7. ETHICAL APPROVAL: Ethical approval for the United Kingdom was granted by Southampton and South West Hampshire Local Research Ethics Committee (B) (ref. 07/H0504/104). Competent authority approval for the UK was granted by the Medicines and Healthcare Products Regulatory Agency. Ethical and competent authority approval was obtained from each local organisation at every research site outside of the UK. Patients who fulfilled the inclusion criteria were given written and verbal information, and informed consent was obtained before enrolment. BODY.5. CONCLUSIONS: This subgroup analysis provides some evidence that the observed increased risk of any AE or diarrhoea due to amoxicillin was not specific, or more pronounced, in any subgroup of patients. In other words, all adult LRTI patients prescribed antibiotics are likely to be at the same risk for any AE or diarrhoea. We can reiterate the conclusion of the previous GRACE trial that the results do not suggest the use of amoxicillin where only little benefit has been observed for patients with uncomplicated LRTI in primary care. Before prescribing an antibiotic, their potential benefits and harms should be discussed with patients.

TITLE: Is Routine Iron Supplementation Necessary in Pregnant Women With High Hemoglobin? ABSTRACT.BACKGROUND:: Iron supplementation is a chief component in prenatal care, with the aim of preventing anemia; however, extreme maternal iron status may adversely affect the birth outcome. Given the negative consequences of high maternal iron concentrations on pregnancy outcomes, it seems that iron supplementation in women with high hemoglobin (Hb) should be limited. ABSTRACT.OBJECTIVES:: The aim of this study was to examine the effect of iron supplementation on iron status markers in pregnant women with high Hb. ABSTRACT.PATIENTS AND METHODS:: In a randomized, double-blind, placebo-controlled trial, 86 pregnant women with Hb > 13.2 g/dL and ferritin > 15 μg/l in the 16th - 20th week of pregnancy were randomized into experimental and control groups. From the 20th week until the end of pregnancy, the experimental group received one ferrous sulfate tablet containing 50 mg of elemental iron daily, while the control group received a placebo. Hb and ferritin levels at 37 - 39 weeks of pregnancy were evaluated and compared. In addition, after delivery the birth weight was measured in two groups and compared. ABSTRACT.RESULTS:: There were statistically significant differences between the two groups in Hb (p = 0/03) and ferritin (p = 0/04) levels at the end of pregnancy, but the incidence of anemia exhibited no difference in either group (p < 0/001). In addition, the mean of birth weight in experimental group and control group were 3391/56 ± 422, 3314/06 ± 341, respectively and it was not significant difference (p = 0.2). ABSTRACT.CONCLUSIONS:: Not using iron supplementation did not cause of anemia in women with Hb concentrations greater than 13.2 g/dL during pregnancy; thus, the systematic care and control of iron status markers without iron supplementation is recommended for these women. BODY.1. BACKGROUND: During pregnancy, the increase in plasma volume exceeds the increase in red cell volume; this causes a physiological hem dilution resulting in reduced hemoglobin (Hb) concentration (1). In a normal pregnancy without iron supplementation, maternal Hb has been found to fall from an average of between 12.5 - 13.0 g/dL to an average of 11.0 - 11.5 g/dL (2). Based on the world health organization (WHO) documents, it is estimated that more than 40% of pregnant women suffer from anemia, which is due to iron deficiency anemia about half of the time (3). Thus, in the recently published guidelines by the WHO, 30 - 60 mg of elemental iron supplementation is advised for all pregnant women (4). However, excessive iron consumption might lead to an increase in Hb levels and blood viscosity (5-10). This condition results in poor placental blood transfusion and adversely affects birth outcome such as preterm delivery, low birth weight (LBW), intra-uterine growth retardation (IUGR), postnatal growth retardation, low fetal head circumference, preeclampsia, and maternal hypertension, as well as fetal and early neonatal death, neurological and skeletal abnormalities, abnormal lung development, and even prenatal mortality (5, 6, 9-12). Moreover, it is clear that oral iron consumption may be unpleasant due to increasing gastrointestinal complications such as nausea, vomiting, abdominal pain. and constipation. In addition, some studies have confirmed that it can reduce intestinal absorption of some trace elements such as zinc and copper (13, 14). Given these considerations, it has been suggested that iron supplementation should not be used for all pregnant women. Instead, it is better to prescribe iron supplementation only if the Hb concentration falls below 10.0 g/dL (14). At the same time, there is still a lot of conflicting information about iron supplementation during pregnancy (15). Considering the negative consequences of extreme maternal iron status on pregnancy outcomes, it seems that iron supplementation in women with high Hb should be limited. BODY.2. OBJECTIVES: Due to complications resulting from iron consumption, we aimed to examine the effect of iron supplementation on maternal iron markers and birth weight in pregnant women with high Hb levels. BODY.3. PATIENTS AND METHODS: Our study was a randomized, double-blind, placebo-controlled clinical trial (RCT registration code: IRCT2013020612383N1) carried out from May 2012 to July 2013 with 86 healthy pregnant women receiving prenatal care at two prenatal clinics in Ardabil, northwest of Iran. These clinics are the only major governmental prenatal clinics in Ardabil, such that most of the pregnant women from different areas of the city are referred to them. The samples were selected from these centers using the convenience sampling method based on ɑ = 0.05, S = 11.17, and d = 2.36 (Equation 1, sample size formula). (1)n=z2.s2d2=(1.96)2×(11.17)2(2.85)2=3.84×124.768.12=64We used high Hb and serum ferritin in pregnancy as the chief criteria for sampling. Participants were all in the 16th–20th week of pregnancy, and all had a Hb concentration greater than 13.2 g/dL and serum ferritin levels above than 15 μg/L (according to the centers for disease control (CDC) and prevention recommendations) (16). Hb concentration and serum ferritin levels were evaluated twice during the study, before the intervention and at end of pregnancy (37 - 39 weeks). To increase the reliability of the laboratory results, all of the blood samples were evaluated in one specific laboratory by one trained person where the laboratory equipment was calibrated. The complete blood count was measured with an automatic cell counter (Hycell, France) and serum ferritin was measured by enzyme-linked immunosorbent assay (ORG5Fe, Bngomtak, Germany). Inclusion criteria were a maternal age of 18 - 35 years, no medical disease, a body mass index (BMI) of 19.8 - 26 kg/m2, an Hb level more than 13.2 g/dL and ferritin level more than 15 μg/L, and having a singleton pregnancy with 16 - 20 weeks gestational age (GA). BMI was calculated as weight in kilograms divided by the square of height in meters (17), while GA was based on a reliable, self-reported estimate of last menstrual period (LMP) or an ultrasound done early in pregnancy if LMP was unknown. When both estimates were available and were within 14 days of one another, we used the LMP to estimate GA. When the difference in estimates exceeded 14 days, we used the ultrasound (1). Exclusion criteria were smoking or having a disease related to polycythemia, such as asthma or chronic hypertension, renal disease, malignancy, or a known blood disorder. To allow for follow-up loss, 86 women were enrolled and randomized. Simple randomization from a table of random numbers was used to assign the women to the iron supplementation group (experimental group) or placebo group (control). The experimental group received one ferrous sulfate tablet containing 50 mg of elemental iron daily, while the control group received a placebo. Because iron supplementation was expected to be necessary after delivery and during the breastfeeding period, and to consider ethical concerns in this regard, all women received 50 mg of elemental iron daily for 3 months after delivery as routine postnatal care. The project was approved by the institutional ethical committee. Informed consent was obtained from all women and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. All mothers were followed up for the evaluation and comparison of changes in Hb and ferritin levels at the end of pregnancy (37 - 39 weeks of gestation). In the case of diagnosed anemia (≤ 11 g/dL) (16), iron supplementation was initiated and continued until delivery. After delivery, infants' weight was measured using Sea scale (accuracy: 10g). During the study (after randomization), 22 participants were excluded for different reasons, such as preterm delivery (n = 5), vaginal bleeding (n = 5), anemia (n = 2), preeclampsia (n = 3), and loss to follow-up (n = 7). Thus, data from 64 of pregnant women were analyzed and rechecked by two researchers. Finally, we used the confirmation of an associated specialist for assurance. In advance of the study, a computer randomization program was used to assign participants to either the intervention or control group. Due to carefully planned follow-up, there were no missing values. Normal distribution was checked using the one-sample Kolmogorov-Smirnov (KS) test. The independent t-test was used to compare variables between the two groups. P < 0.05 was considered significant. BODY.4. RESULTS: During the study of 86 pregnant women, 22 dropped out for different reasons, as mentioned above (Patients and Methods section). Thus, data from 64 pregnant women were used for statistical analysis (Figure 1). Figure 1.BODY.SAMPLING DIAGRAM: The mean maternal age and BMI were 26.11 ± 5.13 and 23.9 ± 2.32, respectively. In addition, 50% of women were nulliparous and 81.3% were housewives. There were no significant difference between the experimental and control groups in terms of maternal age, pre-pregnancy BMI, parity, or Hb and ferritin levels at the onset of the trial (Table 1). After the intervention, at the end of pregnancy, Hb and ferritin levels were determined and compared; also the birth weight was measured and compared after delivery. The mean Hb concentration was 12.05 ± 0.9 in the experimental and 11.94 ± 0.6 in the placebo group; this different was significant (P = 0.03) but there was not significant different between 2 groups in the mean of birth weight (P = 0.2) (; Table 2). Although four pregnant women in both groups were anemic (Hb < 11 g/dL) at the end of pregnancy, none of them had Hb less than 10 g/dL, and this difference was not significant between the iron supplementation and placebo groups. Comparing these results with the CDC criteria to define anemia during the third trimesters of pregnancy, we concluded that anemia had not occurred even though the women had not consumed iron supplementation during pregnancy. In addition, the Hb concentrations of one pregnant woman in the placebo and two pregnant women in the experimental group were greater than 13.2 g/dL; this difference was not significant (Table 3). Table 1.BODY. DEMOGRAPHIC AND OBSTETRIC CHARACTERISTICS OF THE PARTICIPANTS BEFORE THE INTERVENTION:a Iron Supplementation GroupPlacebo GroupP Value Maternal age, y 26.59 ± 5.26 25.63 ± 5.04 0.7 BMI, kg/m 2 24.12 ± 2.17 23.68 ± 2.47 0.1 Parity 1.68 ± 0.8 1.65 ± 0.7 0.2 Hb level, g/dL 13.69 ± 0.44 13.57 ± 0.4 0.2 Ferritin level, μg/L 33.93 ± 13.72 37.05 ± 16.86 0.1 Abbreviations: BMI, body mass building; Hb, hemoglobin. aValues are expressed as mean ± SD. Table 2.BODY.LEVELS OF HEMOGLOBIN AND FERRITIN IN THE IRON SUPPLEMENTATION AND PLACEBO GROUPS AT THE END OF PREGNANCY:a Iron Supplementation GroupPlacebo GroupP Value Hb level, g/dL 12.05 ± 0.9 11.94 ± 0.65 0.03 Ferritin level, μg/L 28.5 ± 9.3 27.22 ± 12.96 0.04 Birth weight 3391/56 ± 422 3314/06 ± 341 0.2 Abbreviations: Hb, hemoglobin. aValues are expressed as mean ± SD. Table 3.BODY. NUMBER OF PREGNANT WOMEN WITH DIFFERENT LEVELS OF HEMOGLOBIN IN THE EXPERIMENTAL AND CONTROL GROUPS AT THE END OF PREGNANCY:a Hb Level, g/dLIron Supplementation GroupPlacebo GroupP Value < 10 0 0 NA 10 - 13.2 31 (96.87) 30 (93.75) NA > 13.2 1 (3.13) 2 (6.25) NA Abbreviations: Hb, hemoglobin. aValues are expressed as No. (%). BODY.5. DISCUSSION: The present study aimed to investigate the effect of iron supplementation on iron status markers and birth weight in pregnant women with high Hb levels. According to some studies, iron status can be predicted among women of childbearing age using the complete blood count test (18), but we also used ferritin level as the best iron status marker to increase the reliability of our study. We found a significant difference between both ferritin and Hb levels in the experimental and control groups at the end of pregnancy. This different was not significant before the intervention. No participants in either the experimental or control group had Hb < 10 g/dL. Previous studies confirmed that Hb greater than 13.2 g/dL increases adverse pregnancy outcomes; thus, we chose this cut-off level as a high level of Hb in pregnancy (2, 19-21). Our findings are in accordance with a report by Steer from 2013 (14). In our study, there were significant differences in Hb and ferritin levels at the end of pregnancy in the iron supplementation group and the placebo group, but the incidence of anemia was similar in both groups, wherein mild anemia was observed in a few women. These findings of this research are in accordance with those of Ziaei et al. (7). Mei et al. revealed that iron supplementation of non-anemic women during pregnancy can affect the iron status later in pregnancy; however, they did not observe this effect during the perinatal period (22). However, Roberfroid et al. claimed that iron supplementation of non-anemic pregnant women might have some benefits. For instance, they stated that even a low dose of iron supplementation of non-anemic mothers during pregnancy can increase children’s birth weight. Moreover, they found that during pregnancy, Hb concentrations decreased even in pregnant women without anemia (23). Although in our study the birth weight in iron supplementation group was more than placebo group, this different was not significant. Perhaps it is needed to study it in the large sample in future. Gonzales et al. conducted a large study in which changes of Hb concentration during pregnancy were measured in 379,816 non-anemic pregnant women. They found that Hb concentrations in most of the non-anemic pregnant women had not changed significant at the end of pregnancy, and moderate/severe anemia was observed only among 2.8% of those women. Furthermore, they revealed that the risk of anemia at the end of pregnancy in non-anemic women diagnosed early during the same pregnancy increased following higher gestational age at the second measurement of Hb, BMI < 19.9 kg/m2, living without a partner, fewer than five antenatal care visits, first parity, multiparty, and preeclampsia (24). A meta-analysis by Haider et al. showed no significant difference of maternal anemia at the time of delivery between mothers who were anemic and non-anemic women during the first trimester (25). In conclusion, our trial suggests that individual iron prophylaxis according to iron status is preferred to routine iron prophylaxis in pregnant women. Since we did not evaluate not using iron supplementation on pregnancy outcomes and the postpartum period, it is suggested that this effect should be investigated in the future studies. Our study had some strengths and limitations. Some studies investigated the correlation of high Hb concentration and pregnancy outcomes; however, to our knowledge, none of these studies have investigated the effect of not using iron supplementation in non-anemic pregnant women on the incidence of anemia near delivery. In addition, none of these articles measured ferritin level at the time of delivery in these women. However, due to the low sample size in this study, we cannot extend our results. Therefore, it is recommended that similar studies should be conducted with a larger sample size in relation to this issue.

TITLE: The Effect of Intraoperative Use of High-Dose Remifentanil on Postoperative Insulin Resistance and Muscle Protein Catabolism: A Randomized Controlled Study Objective: We investigated the effect of the intraoperative use of a high dose remifentanil on insulin resistance and muscle protein catabolism. Design: Randomized controlled study. Patients and Intervention: Thirty-seven patients undergoing elective gastrectomy were randomly assigned to 2 groups that received remifentanil at infusion rates of 0.1 μg·kg-1·min-1 (Group L) and 0.5 μg·kg-1·min-1 (Group H). Main outcome measures: Primary efficacy parameters were changes in homeostasis model assessment as an index of insulin resistance (HOMA-IR) and 3-methylhistidine/creatinine (3-MH/Cr). HOMA-IR was used to evaluate insulin resistance, and 3-MH/Cr was used to evaluate the progress of muscle protein catabolism. Intraoperative stress hormones, insulin, and blood glucose were assessed as secondary endpoints. Results: Eighteen patients in Group L and 19 in Group H were examined. HOMA-IR values varied within normal limits in both groups during surgery, exceeding normal limits at 12 h after surgery and being significantly elevated in Group L. There were no significant differences in the 3-MH/Cr values between the 2 groups at any time point. The stress hormones (adrenocorticotropic hormone, cortisol, and adrenaline) were significantly elevated in Group L at 60 min after the start of surgery and at the initiation of skin closure. There were no significant differences in insulin values, but blood glucose was significantly elevated in Group L at 60 min after the start of surgery and at the start of skin closure. Conclusion: Use of high-dose remifentanil as intraoperative analgesia during elective gastrectomy reduced postoperative insulin resistance, although it did not reduce postoperative muscle protein catabolism. BODY.INTRODUCTION: In recent years, perioperative management methods for enhancing recovery after surgery and improving patient prognosis have been suggested on the basis of evidence from many studies 1. Such management methods are known as “enhanced recovery after surgery programs.” In the programs, measures taken throughout the perioperative period to reduce postoperative insulin resistance are believed to have effects on the enhanced recovery after surgery 2, 3. Although there are a number of indices that influence the capacity for postoperative recovery, poor intraoperative glucose control is a risk factor for the incidence of serious postoperative complications 4 and affects mortality 5. Insulin resistance has been reported to exert a negative effect on postoperative patient outcomes under conditions associated with prolonged hospitalization 6 and postoperative wound infection 7. Poor intraoperative blood glucose control likely results in stress-induced hyperglycemia due to surgical invasion, complicated with protein catabolism in vivo. As previous studies indicates, remifentanil-focused anaesthetic management improves blood glucose control by suppressing intraoperative stress hormone secretion and preventing hyperglycemia 8-10. There have been no investigations, however, on the effect of remifentanil dosage on postoperative insulin resistance and muscle protein catabolism. In this study, we investigated the hypothesis that intraoperative use of high-dose remifentanil may reduce perioperative insulin resistance and muscle protein catabolism by suppressing the stress-induced neuroendocrine response. BODY.METHODS: BODY.1. ETHICS: Ethical approval for this study (Trial No. 26) was provided by the Institutional Review Board of Kanagawa Cancer Center, Japan on September 24, 2010, and the study was carried out in accordance with the 2004 Helsinki Declaration BODY.2. SUBJECTS: Subjects were patients scheduled to undergo laparotomic or laparoscopic elective gastrectomy (total gastrectomy or distal gastrectomy). They met all the following eligibility criteria. The criteria were as follows: Age ≥45 and <75 years, American Society of Anesthesiologists physical status (ASA-PS) grade I or II with body mass index under 35 Kg·m-2 , and written informed consent obtained prior to enrollment. Exclusion criteria were as follows: advanced cancer-related muscle protein catabolism prior to surgery 11, steroid administration, hemoglobin A1c >6.5% , intraoperative blood loss more than 500 mL, and insulin or glucose administrated intraoperatively. Total number of subject enrollment was set to be 46, of which 23 subjects were for the low-dose 0.25 μg·kg-1·min-1 remifentanil anaesthesia group (Group L) and 23 subjects were for the high-dose 0.5 μg·kg-1·min-1 remifentanil anaesthesia group (Group H). As this study was an exploratory investigation, and because there is not the study that investigated relationship between the endpoint and remifentanil consumption in the past, the target sample size was based on the assumption that “blood glucose levels can constitute an endpoint, indirectly expressing comparable levels of analgesia.” Since blood glucose data from previous studies presented blood glucose levels of 118 mg·dL-1 for patients receiving 0.25 μg·kg-1·min-1 remifentanil and 92 mg·dL-1 for those receiving 1.0 μg·kg-1·min-110, average blood glucose levels in this study were set at 120 mg·dL-1 for Group L and 100 mg·dL-1 for Group H. With standard deviations of 20 mg·dL-1 for both groups, a 2-sided significance level of 0.05, and 90% power, it was estimated that the sample size required for a t-test was to be 44 (22 patients per group), and enrollment was therefore set at 46 patients (23 patients per group) to allow for cases of dropouts and withdrawals 12. BODY.3. EXPERIMENTAL PROCEDURE: This study was designed as a randomized controlled trial. Subjects were registered using a patient allocation table by block randomization and allocated to either Group L or Group H. The same preoperative management was given to the patients of both L and H groups 13. Patients fasted after taking an evening meal at 18:00 the night before surgery and were allowed to drink an oral rehydration solution (ORS) up to 2 h before entering the operating room. ORS contains 2.5% carbohydrate and 50 mmol·L-1 sodium ions. Preoperative body fluid management with ORS is known as preoperative oral rehydration therapy (ORT) and is widely practiced in Japan instead of infusion therapy 14,15. In our hospital, patients are asked to drink 1500 mL of ORS packaged in 500-mL plastic bottles (OS-1, Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) for preoperative fluid and electrolyte replacement. After a patient entered the operating room, a 20G intravenous line was secured in the patient's left forearm and a glucose-free infusion of bicarbonated Ringer's solution was administered intravenously. An epidural catheter was then placed between the Th 7-11 vertebrae, and a test injection of 2.0 mL of 1% mepivacaine was administered. After the induction of general anaesthesia by the intravenous administration of 0.5 μg·kg-1·min-1 remifentanil, 1 mg·kg-1 propofol, and 0.5-1.5 mg·kg-1 rocuronium, tracheal intubation was performed. When intubation was difficult and several attempts were required, it was so described in the patient report form. An arterial pressure line was placed in the radial artery of the left arm after induction of general anaesthesia. Except for maintenance rate of remifentanil infusion, general anaesthesia was maintained similarly in the both groups. Epidural analgesia and intravenous fluids containing glucose were not used during surgery. Additional rocuronium was administered as a muscle relaxant to control adductor pollicis contraction to ≤10%. Propofol at around 2 μg·mL-1 or 4 mg·kg-1·h-1 was administered by target-controlled infusion (TCI) as a sedative. The dose of propofol was adjusted using a bispectral index (BIS) monitor (Nihon Kohden Corporation, Tokyo, Japan) with an index BIS value of 45-60. The dose of remifentanil after tracheal intubation was not predetermined, but was fixed 3 min before the operation started at 0.1 μg·kg-1·min-1 for Group L and 0.5 μg·kg-1·min-1 for Group H. Hemodynamic depression was treated by the administration of 8 mg ephedrine or 0.1 mg phenylephrine for decreased SBP, or the administration of 0.5 mg atropine for decreased HR. Stress response was treated by bolus administration of 1 μg·kg-1 remifentanil. If there was no response to this treatment, noradrenaline or β-blocker was used. Remifentanil infusion was terminated after the completion of blood sampling at the start of skin closure, and continuous epidural analgesia was started as postoperative analgesia after intravenous administration of fentanyl 100 μg and flurbiprofen 50 mg, irrespective of body weight, in both groups. Continuous epidural analgesia was initiated with a bolus dose of 3 mL of 0.2% ropivacaine, followed by continuous administration (4 mL·h-1) of a mixture of 0.2% ropivacaine (280 mL) and 1,000 μg fentanyl via a disposable pump (Coopdeck Balloonjector, 300 mL, Daiken Medical Co., Ltd., Tokyo, Japan). After the tracheal tube was removed, continuous administration of a maintenance infusion containing 7.5% glucose was performed at a rate of 60 mL·h-1 for 12 h following surgery. In addition, pain control was performed by bolus administration of epidural anaesthesia or non-steroidal anti-inflammatory drug (NSAID) suppositories as required. BODY.4. ENDPOINTS: The primary endpoint of this study was the assessment of insulin resistance and muscle protein catabolism at 12 h after surgery. Insulin resistance was assessed by homeostasis model assessment as an index of insulin resistance (HOMA-IR), calculated by the equation “[fasting insulin (μU·mL-1) × fasting blood glucose (mg·dL-1)/405].” Its standard value for the Japanese population is <1.6, and values of ≥2.5 indicate insulin resistance 16. We also obtained HOMA-β (%), calculated by the equation “fasting insulin (μU·mL-1) × 360/[fasting blood glucose (mg·dL-1) - 63]” to assess the secretory function of pancreatic β-cells. This was calculated because it was necessary to know whether abnormal values for HOMA-IR and HOMA-β were due to insulin resistance or pancreatic β-cell dysfunction. HOMA-β is expressed as a percentage (the constants are set as such that its value is 100% when β-cells are functioning at maximum ) 17. However, no standard value for the Japanese population has been established 17. Muscle protein catabolism was assessed in terms of changes in blood 3-methylhistidine/creatinine (3-MH/Cr) 18 and transthyretin (TTR), a rapid turnover protein. Intraoperative stress hormones (adrenocorticotropic hormone, ACTH; cortisol; adrenaline, Ad; noradrenaline, NAd; and dopamine, DOA), insulin, and blood glucose were assessed as secondary endpoints. HOMA-IR and 3-MH/Cr were measured before anaesthesia induction, 60 min after the start of surgery, at the start of skin closure, and 12 h after the end of surgery. TTR was measured before anaesthesia induction, 12 h after the end of surgery, and 7 days after surgery. Stress hormones, insulin, and blood glucose were measured before anaesthesia induction, 60 min after the start of surgery, and at the start of skin closure. BODY.5. STATISTICAL ANALYSIS: Values were expressed as mean ± standard deviation. Groups were compared using χ2 test for nominal scales, Wilcoxon paired rank sum test for ordinal scales, repeated-measures analysis of variance (ANOVA), and unpaired Student's t-test for continuous scales, with p < 0.05 regarded as significant. Changes from the baseline (pre surgery) values were different between the groups by using the unpaired Student's t-test. Mean value was higher than at baseline (pre surgery) within the same group by using the repeated-measures of ANOVA, and subsequently, multiple comparisons were performed using the Tukey-Kramer HSD test. The statistical analysis software used was JMP©8.0.1 (SAS Institute, Tokyo, Japan）. BODY.RESULTS: A total of 46 patients were enrolled, 23 in each group. Of these, the study could be completed as planned by 18 patients (78%) in Group L and 19 (83%) in Group H (Figure 1). Patient characteristics and intraoperative variables are given in Table 1. There were no significant differences between the 2 groups in gender, age, height, body weight, ASA-PS, type of surgery, operation time, anaesthesia time, fluid volume, or blood loss. Intraoperative urine volume, however, was higher in Group H. Intraoperative drug consumption of remifentanil, propofol, and rocuronium was calculated by dividing total consumption by administration times and body weight. Remifentanil consumption was higher in Group H, whereas propofol and rocuronium were used to a greater extent in Group L. Figure 2 shows the HOMA-IR and HOMA-β measurements. HOMA-IR varied within normal limits before induction of anaesthesia, 60 min after the start of surgery, and at the start of skin closure, with no significant differences between the 2 groups. However, at 12 h after the end of surgery, mean values exceeded the standard range (<1.6) in both groups and HOMA-IR value was significantly higher in the Group L than in the Group H. HOMA-β values at 60 min after the start of surgery and skin closure were significantly lower in Group L than in the Group H, but significant difference was not noted. It was significantly lower at skin closure compared to baseline value in the Group L. Figure 3 shows 3-MH/Cr and TTR measurements. There were no significant differences in 3-MH/Cr and TTR measurements between the 2 groups at any time point. In both groups, although TTR level was within normal limits (22-44 mg·dL-1) before anaesthesia induction, it decreased below the standard value at 12 h after the end of surgery and at 7 days after surgery. Figure 4 shows stress hormone measurements. The stress hormones ACTH, cortisol, and Ad were significantly elevated in Group L at 60 min after the start of surgery and at the start of skin closure. Figure 5 shows insulin and blood glucose measurements. There were no significant differences in insulin values between the 2 groups during surgery, but blood glucose was significantly elevated in Group L at 60 min after the start of surgery and at the start of skin closure. The frequency of management for intraoperative hemodynamic depression and stress responses is indicated in Figure 6. Vasopressors were used to treat hemodynamic depression more often in Group H, although this difference was not significant, and treatment for stress response due to surgical invasion was significantly frequent in Group L. Patients were not given any medication or fluids in the recovery room other than those scheduled. BODY.DISCUSSION: In this study, we investigated the effect of the intraoperative use of remifentanil at infusion rates of 0.1 μg·kg-1·min-1 (Group L) and 0.5 μg·kg-1·min-1 (Group H) on postoperative insulin resistance and muscle protein catabolism in patients undergoing elective gastrectomy. In addition, in this study, the patients did not consume carbohydrate fluids before surgery 1, 3, and we did not start epidural analgesia 1, 2,19 or administer glucose-containing fluids 20 during surgery. This was in order to assess the effect of different rates of remifentanil infusion alone on insulin resistance and muscle protein catabolism. In the study, we found that secretion of stress hormones during surgery was suppressed in Group H, and postoperative insulin resistance decreased at 12 h after the end of surgery. However, there was no significant difference between the 2 groups in the level of muscle protein catabolism. Secretion of stress hormones and blood glucose elevation were probably suppressed during surgery in Group H as a result of the use of high-dose remifentanil, as shown in previous studies 8-10. Another finding was that urine volume was higher in Group H despite the absence of any difference between the 2 groups in fluid volume, which was consistent with the study of Myles et al 21. HOMA-IR was significantly elevated in Group L at 12 h after the end of surgery. Generally, a highly reliable method such as the glucose clamp test, steady state plasma glucose technique, or minimal model method is chosen for assessing insulin resistance. In this study, however, we chose to use HOMA-IR for the following reasons: involvement of a large number of subjects, necessity to perform assessments with complicated postoperative procedures, and fact that HOMA-IR is recognized as highly reliable in patients with blood glucose ≤140 mg·dL-122. HOMA-β was lower in Group L at 60 min after the start of surgery and at the start of skin closure, although significant difference was not recognized because of large values of standard deviation. These findings suggest that in Group L, the decline in insulin secretion by pancreatic β-cells was maintained during surgery and also suggest that although secretory function recovered after surgery, the response to insulin receptors in cells was poor. This was probably why there was no attenuation of postoperative insulin resistance in Group L. Since HOMA-IR is calculated from the amount of insulin excretion and the level of blood glucose, changes in HOMA-IR are not likely to occur during inoperative periods or at the time of unusual conditions when glucose is not loaded, whereas changes are very likely to occur in postoperative periods when glucose is loaded. In this study, no difference was noted in HOMA-IR values between the groups during and at the end of surgery, although significant difference was noted 12 hr later. We also used blood 3-methylhistidine/creatinine (3-MH/Cr), rather than urine methylhistidine, as an index of muscle protein catabolism. This is because metabolic assessment of 3-MH/Cr is possible with blood in a shorter time than with urine 18. We measured such 3-MH/Cr to assess muscle protein catabolism due to surgical invasion. We initially expected that the use of high-dose remifentanil would give rise to differences in stress-induced muscle protein catabolism. However, for all patients, values varied within normal limits (0.13-0.53 nmol·μg-1 Cr), although the values were close to the upper limit, and there was no significant difference between the 2 groups. 3-MH/Cr may have remained within normal limits in Group L due to the effect of the lipids in propofol which was used as a sedative. Propofol contains an amount of lipid equivalent to a 10% fat preparation. A large dose of propofol is required to guarantee a consistent level of sedation through its interaction with remifentanil, and as a result propofol consumption was significantly larger in Group L compared with that in Group H. It is therefore very likely that because Group L received a larger dose of fat than did Group H, muscle protein catabolism decreased and there was no rise in 3-MH/Cr. The results of this study have shown that strong intraoperative analgesics may suppress the increase of stress hormone and blood glucose level during surgery, and thus affecting insulin resistance after surgery. Our findings in this study suggest that concomitant use of 0.5 μg·kg-1·min-1 remifentanil as an intraoperative analgesic with the aim of improving insulin resistance may offer new possibilities for enhanced recovery after surgery. Regarding the patient enrollment, this study included patients undergoing both open surgery and laparoscopic surgery, which differ in the degree of surgical invasiveness. In the study, stratified randomization was performed for each type of surgery, and there was no bias among patients registered in the 2 groups. We compared data for patients who underwent open surgery and laparoscopic surgery within each group and found no significant difference in variables. This study had several limitations. There were a number of dropout cases (due to operative blood loss, type of surgical procedure applied, etc.) and a planned sample size (22 patients per group) was not investigated in this study. We did not investigate the endpoints, which were used to assess recovery after surgery in other studies, such as length of hospital stay and occurrence of postoperative complications 23, so that we were unable to conclude whether the use of high-dose remifentanil may enhance recovery after surgery. Several previous studies, however, have reported that reducing postoperative insulin resistance enhances recovery after surgery 1-3, 24. We used HOMA-IR to assess insulin resistance. Because the results of this study showed a tendency to reduce insulin resistance in postoperative patients, it will be necessary to confirm these results with greater accuracy by limiting studies to small numbers of patients and using a method such as the glucose clamp test. The results of this study support the hypothesis that the intraoperative use of high-dose remifentanil may reduce postoperative insulin resistance patients undergoing elective gastrectomy. However, the hypothesis that it may reduce postoperative protein catabolism was not established.

TITLE: Aseptically processed and chemically sterilized BTB allografts for anterior cruciate ligament reconstruction: a prospective randomized study ABSTRACT.PURPOSE: To compare the clinical outcomes of bone-patellar tendon-bone (BTB) allografts processed via a novel sterilization system with the traditional aseptically processed BTB allografts for anterior cruciate ligament (ACL) reconstruction. ABSTRACT.METHODS: A total of 67 patients undergoing ACL reconstruction at 6 independent investigation sites were randomized into one of two intervention groups, BioCleanse-sterilized or aseptic BTB allografts. Inclusion criteria included an acute, isolated, unilateral ACL tear, and exclusion criteria included prior ACL injury, multi-ligament reconstruction, and signs of degenerative joint disease. Post-op examiners and patients were blinded to graft type. Patients were evaluated at 6, 12, and 24 months. Clinical outcomes were compared using the IKDC, a KT-1000 knee arthrometer, level of effusion, and ranges of motion (ROM). ABSTRACT.RESULTS: After randomization, 24 patients received aseptic BTB allografts and 43 patients received BioCleanse-sterilized allografts. Significant improvement in IKDC scores (P < 0.0001) as well as KT-1000 results (P < 0.0001) was noted over the 24-month period for both groups. IKDC or KT-1000 results were not significantly different between groups at any time point. Active flexion ROM significantly improved from pre-op to 24-month follow-up (P < 0.0001) with no difference between groups at any time point. Active extension ROM did not differ significantly between the two groups. ABSTRACT.CONCLUSIONS: These results indicate that the sterilization process, BioCleanse, did not demonstrate a statistical difference in clinical outcomes for the BTB allograft at 2 years. The BioCleanse process may provide surgeons with allografts clinically similar to aseptically processed allograft tissue with the benefit of addressing donor-to-recipient disease. ABSTRACT.LEVEL OF EVIDENCE: II. BODY.INTRODUCTION: In anterior cruciate ligament (ACL) reconstruction, the choice of graft has been an area of debate for several years. While there are arguments made that autograft is the gold standard [6, 40], the use of allografts is relatively common with successful outcomes having been well documented in the literature. The use of allograft tissue has been noted to decrease operating time, eliminate donor site morbidity, and increase the tissue available for multi-ligament cases [24, 42]. Clinical studies that evaluated outcomes reported positive results for chronic ligamentous laxity [23]. Subsequently, there have been a number of reports of successful outcomes using allografts in surgery [7, 15, 16]. Further support for the use of allografts can be seen in long-term studies which have reported positive results for patients as long as 10 years after surgery [1, 20, 23]. With the prospect of increased morbidity associated with autograft procedures [2, 12, 43], the rationale for allograft use in ACL surgery seems warranted. One disadvantage which has received a great deal of attention is that disease can be transmitted as a result of the allograft. Use of allograft tissue has historically been associated with HIV and HCV transmission along with bacterial infections, resulting in significant morbidity and mortality [5]. These risks can be minimized by rigorous donor screening, aseptic harvesting techniques, and tissue processing, but the allograft that is only aseptically processed cannot be guaranteed to be free of all viruses or bacterial spores. Historically, multiple sterilization techniques have been used for allograft tissue, most notably gamma irradiation and ethylene oxide. The use of gamma irradiation and ethylene oxide has decreased over time in favour of less destructive and biologically friendly chemical cleaning processes [18, 26, 27, 30, 36, 38, 39, 42]. The BioCleanse® tissue sterilization process (RTI Biologics, Alachua, FL) is a non-thermal combination of mechanical and chemical processes that has been reported to inactivate or remove all sources of infectious disease transmission while not compromising the biomechanical and physiological properties of allograft bone and soft tissue [21, 22, 29, 34]. To date, there has not been a direct comparison of clinical outcomes via a randomized, prospective clinical trial with this type of sterilized allograft. The purpose of this study was to compare the clinical outcomes of patients who underwent ACL reconstruction with BioCleanse BTB allografts to those who received the traditional aseptically, non-irradiated processed BTB allografts. It was hypothesized that patients undergoing ACL reconstruction with BioCleanse BTB allografts would not have statistically or clinically meaningful differences in outcomes from patients who received non-irradiated aseptically processed BTB allografts. BODY.MATERIALS AND METHODS: Patients who presented with an acute (<4 months), isolated ACL rupture were asked to participate in this study. The patients were at one of 6 independent research sites. A total of 67 patients were randomly assigned, to 1 of 2 groups using the ranblock.exe application, and received either the BioCleanse or aseptic BTB allograft. The mean age of all patients was 34 (SD 9) years. Patients requiring multi-ligament reconstruction, moderate to severe concomitant meniscal repair or ACL revision surgery were excluded. This study was approved by the Institutional Review Board (or equivalent) at all the participating sites and all subjects gave informed consent. Aseptic grafts were obtained from one of 3 tissue banks currently providing BTB allografts in the United States. All donors were screened, and all tissues were harvested and processed according to standards set forth by the American Association of Tissue Banks [3]. Each investigational site chose their own supplier for the aseptic grafts. The BioCleanse tissue sterilization system uses a combination of mechanical and chemical processes, working in conjunction with each other. The mechanical component applies oscillating positive and negative pressure in the presence of the chemical agents (including detergents and sterilants), which perfuse the tissue. This combination removes blood and lipids and inactivates or removes pathogenic microorganisms. Repeated rinses throughout the process remove debris, and final rinses remove residual chemicals, leaving the tissue biocompatible [42]. All patients underwent single incision arthroscopic ACL reconstruction under general anaesthesia. A tourniquet was used in every case. All articular damage was noted and recorded using the Outer-bridge classification system. In addition, all meniscal damage was identified, and lesions were treated with partial menisectomy. Following this, a guide system was used that placed the tibial tunnel centre at the anatomical centre of the native ACL. The femoral tunnel was drilled just anterior to the over the top position at the ‘2AM/10PM’ isometric single bundle location using the standard transtibial approach. An endoscopic femoral aimer was used to minimize patient to patient variability. The grafts were reconstituted using room temperature saline for a minimum of 10 min. The BioCleanse grafts were preshaped with a 10 mm diameter and 25–30 mm length bone blocks. The aseptic grafts were shaped individually in each centre during surgery to the same dimensions as the BioCleanse preshaped grafts. The graft was then pulled distal to proximal via the tibial tunnels. Both femoral and tibial fixations were achieved using metallic interference screws. After fixation at the femur, the graft was tensioned with approximately 20 lbs of force for tibial fixation at near extension. Following this, the knee was put through a full range of motion to verify that there was no graft impingement. Stability was then checked to make sure that both the anterior drawer sign and the pivot shift were eliminated. At this point, the arthroscopic instruments were removed and the distal tibial incision was closed in a standard fashion. All patients followed a uniform ACL rehabilitation protocol. Following surgery, the patients began a therapist-directed physical therapy programme. Post-operative rehabilitation programmes emphasized range of motion restoration, quadriceps strengthening, and patellofemoral joint protection. These were divided into 6 phases using objective criteria for advancement to the next phase; these phases took up to 6 months to complete. At 3, 6, 12, and 24 months post-operatively, the patients returned to their respective clinics for an evaluation including the International Knee Documentation Committee (IKDC) form along with a physical examination for range of motion (ROM) effusion and a KT-1000 knee arthrometer test. All sites received a training DVD demonstrating KT-1000 testing to ensure standard and consistent use when evaluating the ACL at maximum manual testing. Previous research has shown KT-1000 testing to be reliable and repeatable [14, 25, 28]. Both the examiner and the patient were blinded as to the type of graft implanted. BODY.STATISTICAL ANALYSIS: The IKDC scores, KT-1000 measures, and ROM were compared using a repeated-measures ANOVA. A chi-square test and Fisher’s exact test were used to compare between categorical variables, that is, gender distribution and grade of effusion. Demographic characteristics were compared using a Student’s t test. An a priori power analysis was primarily based on KT-1000 measures. Using G3 software (Heinrich Heine University Düsseldorf, Germany) with an effect size of 0.33, alpha of 0.05, power of 0.80, for 3 groups (aseptic, gamma-irradiated, and BioCleanse grafts), we estimated that 112 patients would be required. Due to circumstances such as surgeon preference and patient recruitment, the gamma-irradiated BTB allograft group was dropped. This left the aseptic and BioCleanse grafts for the final analysis. The initial statistical plan had been an unbalanced design, allowing for an unequal number of patients in each group. The level of significance for all statistical tests was set at an alpha level of 0.05. BODY.RESULTS: As shown in Table 1, there was no significant difference between the groups for age, body mass index, or gender distribution. Of the 43 patients enrolled in the BioCleanse group, there were 29, 24, 20, and 18 tested at 3, 6, 12, and 24 months, respectively. For the aseptic group, testing was completed on 23, 20, 13, and 10 patients at 3, 6, 12, and 24 months, respectively.Table 1Descriptive data for the patients in this study Aseptic allograftsSterilized allografts P valueAge (years) 31.3 ± 9.2 35.6 ± 8.9 ns Body mass index 26.7 ± 4.8 26.8 ± 4.7 ns Revision surgeries 1 1 ns Male (n) 16 25 ns Female (n) 8 18 The data for IKDC scores are presented in Fig. 1. The mean IKDC pre-operatively was 51 (95 % CI 42.3–53.8) and 48 (95 % CI 42.3–53.8) for the aseptic and BioCleanse groups, respectively. This improved over the 24 months to 89 (95 % CI 81.2–96.4) for the aseptic group and 88 (95 % CI 80.6–95.4) for the BioCleanse group. There was no statistically significant difference in IKDC scores at any time point measured.Fig. 1The IKDC scores for the 2 groups. There were no statistically significant differences between the 2 groups at any time point Similarly, the KT-1000 measurements steadily improved over time, from a mean of 4.6 mm (95 % CI 3.7–6.1) and 4.3 mm (95 % CI 3.3–5.3) for the aseptic and BioCleanse groups, respectively. At the end of the study, the anterior knee displacement as measured by the KT-1000 was recorded at 1.6 mm (95 % CI 1.1–2.1) and 1.5 mm (95 % CI 0.9–2.1) for aseptic and BioCleanse groups, respectively (Fig. 2). No statistically significant difference in KT-1000 scores was found between the groups at any time point.Fig. 2The mean KT-1000 side-to-side differences for anterior displacement at four time points. No significant differences were found between groups The results for range of motion (ROM) showed no significant differences between groups at any time point. This was true for the active flexion ROM (Fig. 3) as well as the deficit for passive extension, measured between ipsilateral and contralateral legs. Effusion grading showed no significant differences between the groups. There were no complications noted at 24 months.Fig. 3Mean active flexion range of motion for the treated knee for both groups There were no cases of either disease transmission or infection that could be attributed to the grafts. This was true for both aseptic and BioCleanse grafts. BODY.DISCUSSION: This prospectively evaluated clinical outcomes study compared aseptically processed allografts to those treated through the BioCleanse sterilization process. While there have been reports comparing outcomes of irradiated allografts to autografts as well as aseptic allografts, the functional outcomes using BTB grafts that have been chemically, non-thermally sterilized have not been reported in the literature. It should be noted that ‘aseptic’ processing does not necessarily mean that the tissue is free of viruses, bacteria, and/or spores. The possibility of contamination coming from the donor cannot be eliminated and could be inherent to the graft [4], which is why tissue banks attempt to clean with different chemical processes. A number of clinical studies have been published [13, 15, 19, 22, 32, 35, 37, 38] which compared allografts and autografts in ACL reconstruction. A recent meta-analysis demonstrated no clinical difference when Level I studies were analysed [11]. This study attempted a standardized single bundle, isocentric, anatomical footprint ACL reconstruction for each surgical site to try and ensure consistency within the study. These results show comparable outcomes between aseptically processed allografts and those that have been sterilized using the BioCleanse process. The importance of this lies in the reduction in the risk of disease transmission which has been documented [5]. Although appropriate donor screening and aseptic harvesting techniques can reduce the risk [17, 33], a method of sterilization that does not damage the structural integrity of the graft is essential. The BioCleanse process inactivates or removes sources of infectious disease transmission without compromising structural integrity of allograft bone and soft tissue [29, 34]. Validation tests of the process have indicated a sterility assurance level of 10−6 [18]. The FDA also states that a sterility assurance level of 10–6 is necessary for all devices unless there is substantial justification why this level cannot be achieved [43]. An important difference between BioCleanse and other sterilization processes is that BioCleanse does not use irradiation, ethylene oxide, or excessive heat, all of which may adversely impact the properties of the graft tissue [8–10, 26, 36, 41]. There are some limitations to this study. While it may be pointed out that the number of sites may affect reliability, previous literature has pointed to the high intertester reliability when using the KT-1000 [25], and that examiner experience can improve the precision and reliability when using multiple sites [31]. Considering the years of experience of the principal investigators, we expect that the reliability between sites would be high. Statistically, an improved power would make the results more certain. Although 67 patients were initially enrolled in this study, the loss to follow-up resulted in only 18 patients in the BioCleanse group and 10 in the aseptic group at 24 months, which reduced the statistical power in the study. The difference in group sizes had been anticipated in the initial statistical plan for an unbalanced design, yet the diminishing numbers did impact power. It should be noted that the loss of patients to follow-up did affect the power of the final statistical analysis, with the power of the KT analysis calculated to be 0.75, while the power of the IKDC test was calculated to be 0.52. Also, although the same surgical technique and rehabilitation protocol were applied for all patients at all sites, the role of individual technique as well as adherence to rehabilitation guidelines can introduce variance in the results. Lastly, while we had initially attempted to compare BioCleanse, aseptic, and gamma-irradiated allografts, extremely low enrolment in the gamma-irradiated arm prevented an adequate statistical comparison. This was due to a general shift away from irradiated allografts and was coupled with several patients who declined to participate in this study when they were informed they might receive an irradiated graft. BODY.CONCLUSIONS: All patients, whether receiving BioCleanse BTB allografts or aseptically processed BTB allografts, exhibited similar clinical outcomes. There were no complications, with only one revision required in each group. This short-term data shows that the BTB allografts processed through the BioCleanse process provide a viable option for ACL reconstruction while minimizing the risk of disease transmission.

TITLE: Zhibitai and low-dose atorvastatin reduce blood lipids and inflammation in patients with coronary artery disease ABSTRACT.ABSTRACT: ABSTRACT.BACKGROUND:: Atorvastatin decreases blood lipids but is associated with side effects. Zhibitai is a traditional Chinese medicine used to treat blood lipid disorders. The objective of this study is to evaluate the lipid-lowering effect, antiinflammatory effect, and adverse events of zhibitai combined to atorvastatin in patients with coronary heart diseases (CHDs). ABSTRACT.METHODS:: Patients with CHD (n = 150) were randomized to: zhibitai 480 mg + atorvastatin 10 mg (ZA10 group), atorvastatin 20 mg (A20 group), and atorvastatin 40 mg (A40 group). Lipid profile, cardiotrophin-1 (CT-1), and C-reactive protein (CRP) were measured after 4 and 8 weeks of treatment. Self-reported side effects, liver function, kidney function, and creatine kinase levels were monitored. ABSTRACT.RESULTS:: After 8 weeks, triglycerides, total cholesterol (TC), LDL-cholesterol (LDL-C), and apolipoprotein B100 (ApoB100) levels were decreased in the ZA10 group (−64%, −37%, −46%, and −54%, respectively, compared with baseline), and these changes were similar to those of the A40 group (P > 0.05). CT-1 and high sensitivity-C reactive protein (hs-CRP) levels were significantly decreased in the ZA10 group after 4 and 8 weeks (4 weeks: −73% and 96%; 8 weeks: −89% and −98%; all P < 0.01), without differences among the 3 groups (P > 0.05). After 8 weeks of treatment, adverse events (abdominal distention, nausea, vomiting, and hunger) were found in 4, 5, and 7 patients in the ZA10, A20, and A40 groups, respectively. ABSTRACT.CONCLUSION:: ZA10 significantly reduced triglycerides, TC, LDL-C, ApoB, CT-1, and hs-CRP levels in patients with CHD, similar to the effects of A40 and A20, but ZA10 lead to fewer adverse events. 1BODY.INTRODUCTION: Health is the most important construct of our life. The 21st century enabled us to live in improved living conditions. However, although our health status changed, many chronic stages of diseases were integrated into our lives.[1] Physical inactivity is one of the risk factors of atherosclerosis and obesity,[2] and may even be the most important. There is now overwhelming evidence that regular physical activity has important and wide-ranging health benefits. These range from reduced risk of chronic diseases such as heart disease, type 2 diabetes, and some cancers to enhance function and preservation of function with age.[3] Existing research shows that proper physical exercise, a healthy environment and balanced nutrition,[4] and good state of mind is an effective factor for the prevention of coronary heart disease (CHD). Atherosclerosis leads to the narrowing of the lumen of coronary arteries. Eventually, progressive plaque thickening and/or rupture may lead to angina and/or myocardial infarction (MI).[5–8] In the United States, the prevalence of CHD is about 6.2% in people ≥20 years old.[9] The rates of major cardiovascular events are higher in developing countries compared with developed ones.[10] Mortality from ischemic heart disease is the leading cause of mortality worldwide, with 12.7% of the total mortality in 2008.[11] Hyperlipidemia is a major risk factor for the development and progression of atherosclerosis.[5–8] Statins are 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors; this enzyme normally catalyzes the rate-limiting step of cholesterol synthesis.[12] Therefore, statins induce cholesterol depletion within the hepatocytes, leading to the upregulation of the low-density lipoprotein (LDL) receptor in order to obtain cholesterol from blood LDL particles.[13] Statins also decrease C-reactive protein (CRP) levels, but their effect on inflammation is not consistent.[14] Statins are the drugs of choice for patients at risk of CHD or CHD progression.[15] Statins significantly reduce the incidence of all-cause mortality and major coronary events as compared to control in both secondary and primary prevention. Atorvastatin was significantly more effective than pravastatin (OR 0.65, 95% CI 0.43–0.99) and simvastatin (OR 0.68, 95% CI 0.38–0.98) for secondary prevention of major coronary events.[16] Atorvastatin is a 3rd-generation statin with a good efficacy and adequate safety profile.[17] Nevertheless, statins are associated with adverse effects such as myopathy, hepatic toxicity, hyperglycemia, and impaired steroid production; rhabdomyolysis and death are also possible.[18] The risk of adverse effects increases with the higher doses.[19] Statins have been shown to reduce the risk of all-cause mortality among individuals with clinical history of CHD. However, it remains uncertain whether statins have similar mortality benefit in a high-risk primary prevention setting. One literature-based meta-analysis did not find evidence for the benefit of statin therapy on all-cause mortality in a high-risk primary prevention set-up.[20] The findings suggest that intensive dose atorvastatin therapy does not attenuate the progression of coronary calcification compared with standard dose atorvastatin therapy over 12 months.[21] Chinese medicine has been regarded as a kind of complementary therapies in the Western countries. However, it has been one of the mainstream therapies in some Asian countries, such as Taiwan, Korea, and China, including acupuncture, moxibustion, Chinese orthopedics and traumatology, and Chinese herbal medicine (CHM).[22] CHM has a long history and is widely used in the prevention and treatment of a variety of diseases worldwide. CHM has been used to treat stroke for thousands of years. The findings suggest that bioactive components of CHM may improve neurological function, reduce infarct volume, and promote endogenous neurogenesis, including proliferation, migration, and differentiation of neural stem cells after ischemic stroke.[23] Zhibitai is a traditional Chinese medicine that is made of naturally occurring statins and it is used to treat blood lipid disorders. Previous studies have shown that zhibitai was comparable to atorvastatin in terms of lipid-lowering and antiinflammatory effects.[24–26] Therefore, zhibitai could be an appropriate alternative to chemical lipid-lowering drugs. Therefore, the aim of the present study was to further evaluate the lipid-lowering effect, antiinflammatory effect, and adverse events of zhibitai combined to atorvastatin 10 mg in patients with CHDs, and to investigate the advantages of the combination compared with higher doses of atorvastatin. 2BODY.METHODS: 2.1BODY.PATIENTS: This was a prospective study of patients with CHD treated at the Inpatient and Outpatient Departments of the Second Xiangya Hospital of Central South University and at the Peace Hospital Affiliated to Changzhi Medical College (Shanxi, China) between October 2010 and May 2011. The study was approved by the ethical committees of the 2 participating hospitals. All patients signed an informed consent form. The inclusion criteria were: diagnosis of CHD with signs and symptoms of MI (typical chest pains and pain-related ST-T changes; typical chest pains and positive treadmill exercise test; or typical chest pains and coronary angiography showing one or multiple coronary branches with stenosis >50%); >20 but <75 years of age; and volunteered to participate and signed the informed consent form. The exclusion criteria were: history of MI during the past 3 months; history of cerebrovascular accidents during the past 6 months; history of severe trauma or major surgery; nephrotic syndrome; hypothyroidism; any liver or gallbladder disorders; familial hypercholesterolemia; or secondary hyperlipidemia. The following drugs were allowed: angiotensin-converting enzyme inhibitor, angiotensin II receptor blockers, calcium channel blockers, clopidogrel, bayaspirin, and β-receptor blockers, as well as other antiplatelet, anticoagulation, antihypertensive, hypoglycemic, and coronary artery dilation drugs such as syncon. 2.2BODY.STUDY DESIGN: This was a randomized, parallel study. Patients were randomized using a random number table and sealed envelopes prepared by a statistician. Patients in the zhibitai 480 mg + atorvastatin 10 mg (ZA10) group received zhibitai (480 mg, twice daily, batch #WS3–119(Z-119)-2004(Z), Chengdu Di’ao Pharmaceutical Group Corporation, China) and atorvastatin (10 mg, once daily Pfizer Pharmaceutical Co., Ltd.) at night for 8 weeks. The patients in the atorvastatin 20 mg (A20) group received 20 mg of atorvastatin once daily at night for 8 weeks. The patients in the atorvastatin 40 mg (A40) group received 40 mg of atorvastatin once daily at night for 8 weeks. 2.3BODY.TESTING: Fasting peripheral venous blood was collected in the morning before treatment and after 4 and 8 weeks of treatment. Levels of cardiotrophin-1 (CT-1) (Xinqidi Biological technology Co., Ltd., Wuhan, China), high sensitivity-C reactive protein (hs-CRP, P800, Roche Diagnostics, Germany), triglycerides, total cholesterol (TC), LDL-cholesterol (LDL-C), high-density lipoprotein cholesterol, apoprotein B100 (ApoB100), apo A1, creatine kinase, and markers of liver and renal functions were detected using DXI800 and AU5800 automatic biochemical analyzers (Beckman, United States of America). Twelve-lead ECG was performed routinely. 2.4BODY.STATISTICAL ANALYSES: Based on previous studies of zhibitai,[24–26] it was determined that a sample size of 35 per group was necessary to detect a difference at a power of 80% and α = 0.05. SPSS 16.0 (IBM, Armonk, NY) was used for statistical analysis. Continuous data were presented as means ± standard deviation and analyzed using independent t-tests for comparisons between 2 groups or paired t-tests for comparisons before/after treatment. One-way analysis of variance was used for comparison among the 3 groups, with the Tukey post hoc test. Categorical data were presented using frequencies and percentages and analyzed using the chi-square test. All analyses were two-sided and P-values <0.05 were considered statistically significant. 3BODY.RESULTS: 3.1BODY.CHARACTERISTICS OF THE PATIENTS: A total of 150 patients were randomized (n = 50/group). 1 patient in the ZA10 group was lost to follow-up and 1 patient was excluded because of poor compliance. 2 and 3 patients in the A20 and A40 groups, respectively, were excluded because of poor compliance. Therefore, 48, 48, and 47 patients in the ZA10, A20, and A40 groups, respectively, were included in for the statistical analysis. There were no significant differences in age, sex, body mass index, smoking history, liver function, renal function, blood lipids, creatine kinase, blood pressure, diabetes, family history of CHD, and drugs among the 3 groups at baseline (all P > 0.05) (Table 1). Table 1Characteristics of the CHD patients. 3.2BODY.CHANGES IN LIPID PROFILE AFTER TREATMENT: Compared with baseline, blood lipids after 4 and 8 weeks of treatment were significantly changed in all 3 groups. TC, LDL-C, and ApoB100 were decreased significantly compared with baseline (P < 0.01), and the resulting levels were similar among all 3 groups (P > 0.05 among groups at 4 and 8 weeks). The decreases in TG levels were significant in the ZA10 and A40 groups at 4 and 8 weeks (P < 0.01), while the decrease in TG in the A20 group was only significant at 8 weeks (P < 0.01). There were no differences in TG levels among the 3 groups at 8 weeks (P > 0.05) (Table 2). Table 2Changes in lipid profile after zhibitai + atorvastatin or atorvastatin treatment. 3.3BODY.CHANGES IN CT-1 AND HS-CRP AFTER TREATMENT: CT-1 levels were decreased significantly after 4 and 8 weeks of treatment in all 3 groups compared with baseline (P < 0.01), and the resulting levels were similar among all 3 groups (P > 0.05). hs-CRP levels were also decreased significantly after 4 and 8 weeks in the 3 groups compared with baseline (P < 0.05), without difference among the 3 groups (P > 0.05) (Table 3). Table 3Changes in CT-1 and hs-CRP levels after zhibitai + atorvastatin or atorvastatin treatment. 3.4BODY.LIVER AND RENAL FUNCTIONS, CREATINE KINASE, AND ADVERSE REACTIONS: After 8 weeks of treatment, adverse events (abdominal distention, nausea, vomiting, and hunger) were reported in 4, 5, and 7 patients in the ZA10, A20, and A40 groups, respectively. Before treatment, and after 4 weeks, 8 weeks treatment, incidence of creatine kinase increasing, the incidence of myopathy, the incidence of gastrointestinal adverse reactions have no significant difference in the three groups (all P > 0.05), and between the each 2 groups, no significant difference can be observed (P > 0.05), There was no significant difference in CK between the three groups before and after treatment (Table 4). Table 4Adverse events during zhibitai + atorvastatin or atorvastatin treatment. 4BODY.DISCUSSION: Abnormal lipid metabolism is a major cause and an independent risk factor of CHD. Therefore, lipid-lowering therapy is crucial to the treatment and prevention of CHD.[27,28] Statins are the main currently accepted lipid-lowering treatment.[13–15] However, most intensive lipid-lowering studies tried to achieve better clinical efficacy by increasing the doses of statins, which led to increased incidence of adverse effects.[19] Therefore, the aim of the present study was to evaluate the lipid-lowering effect, antiinflammatory effect, and adverse events of zhibitai combined to atorvastatin 10 mg in patients with CHD. The results showed that TG, TC, LDL-C, and ApoB100 levels were decreased in the ZA10 group to a similar extent than in the A40 group. The levels of CT-1 and hs-CRP were significantly decreased in the ZA10 group after 4 and 8 weeks, without difference among the 3 groups. The incidence of adverse events (abdominal distention, nausea, vomiting, and hunger) in the ZA10 group was lower compared with the A20 and A40 groups. Previous studies have shown that zhibitai used alone could lower blood lipids and inflammatory factors (including hs-CRP and CT-1).[24–26] The present study showed that the combined use of zhibitai and atorvastatin 10 mg could achieve a clinical efficacy comparable to that of higher doses of atorvastatin, without additional adverse effects. In the present study, the extent of TG lowering was higher than that of TC lowering when using zhibitai. As the majority of Asian patients with hyperlipidemia are mainly with increased TG,[29] zhibitai could be more appropriate for patients with mixed hyperlipidemia characterized by increased TG. In addition, zhibitai combined to atorvastatin could achieve even better lipid-lowering effects. Therefore, technically, zhibitai could be suitable for Chinese patients with hyperlipidemia. Recent studies have shown that increased serum hs-CRP levels are closely associated with CHD and can be used to evaluate the patients’ condition and stability.[30–32] CT-1 is a member of the IL-6 family.[33] Many recent studies have demonstrated that CT-1 is closely associated with the development and progression of CHD.[34] Increased CT-1 levels are found in patients with CHD and are associated with disease severity. The present study showed that the combined use of zhibitai and atorvastatin significantly reduced CT-1 and hs-CRP levels in patients with CHD. In the present study, there were no significant increases in CK, myopathy, and distinct liver and renal damage in the ZA10 group compared with the A20 or A40 groups, but the number of events was low, preventing any reliable statistical analyses. Nevertheless, previous studies of zhibitai showed a low occurrence of adverse effects.[24–26] The present study is not without limitations. Indeed, the sample size was small and from only 2 hospitals. There were no differences among the groups at 8 weeks, and a noninferiority trial should be performed to confirm this equivalence. Further multicenter prospective trials are necessary to reach firm conclusions about the value of zhibitai for the treatment of hyperlipidemia. In conclusion, ZA10 in patients with CHD significantly reduced TG, TC, LDL-C, and ApoB levels, as well as the inflammatory cytokines CT-1 and hs-CRP. These changes were similar to those achieved using A40 and A20, but ZA10 led to fewer adverse events. 5BODY.LIMITATIONS: As the number of cases in this study is small, the observation time is not long enough, so the data and results are one-sided. This will be improved in future research work. The study is only a 1st sign of possible positive influence of herbal therapy, and further studies in the future are necessary to show positive effects on mortality and cardiac events like myocardial infarction or cardiac sudden death. This is the focus of our study work in the future. BODY.ACKNOWLEDGMENTS: The authors thank National Natural Science Foundation of China (No. 81672264, 81372117) for the support.

TITLE: Long‐Term Outcomes in Belatacept‐ Versus Cyclosporine‐Treated Recipients of Extended Criteria Donor Kidneys: Final Results From BENEFIT‐EXT, a Phase III Randomized StudyDurrbach et al In the Belatacept Evaluation of Nephroprotection and Efficacy as First‐Line Immunosuppression Trial–Extended Criteria Donors (BENEFIT‐EXT), extended criteria donor kidney recipients were randomized to receive belatacept‐based (more intense [MI] or less intense [LI]) or cyclosporine‐based immunosuppression. In prior analyses, belatacept was associated with significantly better renal function compared with cyclosporine. In this prospective analysis of the intent‐to‐treat population, efficacy and safety were compared across regimens at 7 years after transplant. Overall, 128 of 184 belatacept MI–treated, 138 of 175 belatacept LI–treated and 108 of 184 cyclosporine‐treated patients contributed data to these analyses. Hazard ratios (HRs) comparing time to death or graft loss were 0.915 (95% confidence interval [CI] 0.625–1.339; p = 0.65) for belatacept MI versus cyclosporine and 0.927 (95% CI 0.634–1.356; p = 0.70) for belatacept LI versus cyclosporine. Mean estimated GFR (eGFR) plus or minus standard error at 7 years was 53.9 ± 1.9, 54.2 ± 1.9, and 35.3 ± 2.0 mL/min per 1.73 m2 for belatacept MI, belatacept LI and cyclosporine, respectively (p < 0.001 for overall treatment effect). HRs comparing freedom from death, graft loss or eGFR <20 mL/min per 1.73 m2 were 0.754 (95% CI 0.536–1.061; p = 0.10) for belatacept MI versus cyclosporine and 0.706 (95% CI 0.499–0.998; p = 0.05) for belatacept LI versus cyclosporine. Acute rejection rates and safety profiles of belatacept‐ and cyclosporine‐based treatment were similar. De novo donor‐specific antibody incidence was lower for belatacept (p ≤ 0.0001). Relative to cyclosporine, belatacept was associated with similar death and graft loss and improved renal function at 7 years after transplant and had a safety profile consistent with previous reports. In patients transplanted with an extended donation criteria kidney, belatacept‐based immunosuppression is associated with a similar death/graft loss and improved renal function at 7 years posttransplant as a cyclosporine‐based immunosuppression, with a safety profile consistent with previous reports. BODY.ABBREVIATIONS: AEadverse event CIconfidence interval CsAcyclosporine DSAdonor‐specific antibody EBVEpstein–Barr virus ECDexpanded criteria donor eGFRestimated GFR HRhazard ratio KDPIKidney Donor Profile Index LIless intense MImore intense PTLDposttransplant lymphoproliferative disorder q2wevery 2 weeks q4wevery 4 weeks UNOSUnited Network for Organ Sharing BODY.INTRODUCTION: The disparity between the number of patients awaiting kidney transplantation and the number of available donor kidneys continues to increase 1, 2. To address this growing demand, expanded criteria donor (ECD) kidneys are increasingly being used 2, 3; however, compared with recipients of non‐ECD kidneys, those who receive ECD kidneys are at increased risk of graft failure and cardiovascular events and have worse renal function and decreased life expectancy 3, 4, 5, 6, 7, 8, 9, 10. Causes of graft lost include inherited donor lesions 11, the adverse effects of immunosuppression and antibody‐mediated chronic rejection. The calcineurin inhibitors cyclosporine (CsA) and tacrolimus—the existing standard of care for maintenance immunosuppression—are potentially nephrotoxic, which may contribute to declining renal function, the development of chronic allograft nephropathy and graft loss 12, 13, 14, 15, 16, 17, 18. Because ECD kidneys may be more susceptible to the nephrotoxic effects of calcineurin inhibitors, the use of CsA or tacrolimus is of greater concern in recipients of ECD versus non‐ECD kidneys 19. The de novo development of donor‐specific antibodies (DSAs) and patient nonadherence to prescribed immunosuppressive regimens have also been recognized as major risk factors for graft loss 20, 21. Belatacept is a soluble fusion protein composed of a modified version of the extracellular domain of cytotoxic T lymphocyte antigen 4 linked to the Fc domain of a human IgG1 antibody 22. Belatacept selectively inhibits T cell activation through costimulation blockade 23, 24, 25, 26, 27. In 2011, belatacept was approved in the United States and the European Union based in part on 3‐year data from two phase III trials: Belatacept Evaluation of Nephroprotection and Efficacy as First‐Line Immunosuppression Trial (BENEFIT) and BENEFIT–Extended Criteria Donors (BENEFIT‐EXT). These randomized phase III studies compared two belatacept‐based immunosuppressive regimens (more intense [MI] and less intense [LI]) with CsA‐based immunosuppression in adult kidney transplant recipients. In BENEFIT‐EXT, analyses performed at 1, 3 and 5 years after transplant demonstrated that belatacept‐based immunosuppression was associated with similar rates of patient and graft survival and superior renal function versus CsA‐based immunosuppression; however, rates of acute rejection were numerically higher with belatacept‐based treatment 28, 29, 30. This report summarizes efficacy and safety outcomes from randomization to year 7 (month 84) in the intent‐to‐treat population of BENEFIT‐EXT. BODY.METHODS: BODY.STUDY DESIGN: The study design of BENEFIT‐EXT (ClinicalTrials.gov identifier NCT00114777) has been described 28. Briefly, this was a 3‐year, international, multicenter, randomized, partially blinded, active‐controlled, parallel‐group study of adults transplanted with an extended criteria donor kidney. Extended criteria donor kidneys were protocol defined as those from donors aged ≥60 years, from donors aged 50–59 years with at least two other risk factors (death due to cerebrovascular accident, history of hypertension, or terminal serum creatinine level >1.5 mg/dL), from donors with an anticipated cold ischemia time ≥24 h, or from non–heart‐beating donors (i.e. donation after cardiac death). Patients were randomized (1:1:1) to receive primary immunosuppression with a belatacept MI–based, belatacept LI–based or CsA‐based regimen. All patients received basiliximab induction, mycophenolate mofetil and corticosteroids. BENEFIT‐EXT was conducted in accordance with the principles outlined in the Declaration of Helsinki. The institutional review board or ethics committee at each site approved the protocol, and all participants provided written informed consent. BODY.OUTCOMES: Efficacy and safety outcomes from randomization to month 84 (year 7), including time to death and/or graft loss, acute rejection, renal function, safety and de novo DSA incidence, are summarized. As in prior analyses 28, 29, 30, acute rejection was defined as central biopsy–proven rejection that was either clinically suspected for protocol‐defined reasons or clinically suspected for other reasons and treated. A combined end point comprising time to first occurrence of death, graft loss or estimated GFR (eGFR) <20 mL/min per 1.73 m2 was examined post hoc. GFR was estimated using the six‐variable MDRD equation 31. Adverse events (AEs) were mapped to terms from the Medical Dictionary for Regulatory Activities version 17.0 (MedDRA MSSO, McLean, VA) and expressed as incidence rates adjusted per 100 person‐years of exposure to assigned treatment. Serious AEs are defined in the supplementary material. De novo DSA development was assessed centrally by solid‐phase flow cytometry (FLowPRA; One Lambda, Inc., Canoga Park, CA), with HLA class specificity (class I or II) determined by LABScreen single antigen beads (One Lambda, Inc.). BODY.STATISTICAL METHODS: For this prospective analysis, time to death or graft loss was compared between each belatacept‐based regimen and the CsA‐based regimen using a log‐rank test. Data are presented using Kaplan–Meier curves and event rates. Cox regression was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for the first 60 and 84 mo. Time to death and time to death‐censored graft loss were assessed as sensitivity analyses to understand the contribution of each individual component to the composite end point of time to death or graft loss; the same statistical methods were used as for the composite analysis, and no adjustment for multiplicity was made. See the supplementary material for censoring rules. Mean eGFR and corresponding CIs were determined from months 1–84 using a repeated‐measures model with an unstructured covariance matrix. This model takes into account between‐subject variability and the intrasubject correlation between eGFR measurements across all time points and assumes that missing data are missing at random. The model included treatment, time and a time–treatment interaction; no further adjustment was made for other potentially confounding covariates. Time was regarded as a categorical variable (intervals of 3 mo up to month 36 and intervals of 6 mo thereafter). A sensitivity analysis was performed in which GFR values that were missing due to death or graft loss were imputed as zero. For this sensitivity analysis, the same model was used as for the primary analysis, but a Toeplitz covariance matrix best fit the data because the unstructured covariance matrix was not converging. A slope‐based model without imputation was also used to determine whether there was a difference between each of the belatacept slopes and the CsA slope, assuming linearity of the eGFR values between months 1 and 84. The difference between slopes was tested using contrasts. Time was regarded as a continuous variable, treatment as a fixed effect, and intercept and time as random effects; no further adjustment was made for other potentially confounding covariates. A sensitivity analysis was performed in which GFR values that were missing due to death or graft loss were imputed as zero; the same model was used as for the slope analysis without imputation. The statistical approaches used in this 7‐year analysis differ from those used at 1, 3, and 5 years after transplant 28, 29, 30. First, in the present report, all evaluable patients were analyzed per the intent‐to‐treat principle; evaluable patients were alive and observable at 84 mo after randomization or died or experienced graft loss by month 84. Similarly, the intent‐to‐treat population was analyzed at 1 and 3 years after transplant 28, 29, whereas a subgroup of patients corresponding to the long‐term extension cohort was analyzed at 5 years after transplant 30. The long‐term extension cohort represented a subset of the intent‐to‐treat population because it was composed of only those patients who completed 36 mo of study treatment and consented to continue the study beyond month 36 30. Second, rates of death and/or graft loss were presented as point estimates at 1 and 3 years after transplant 28, 29, but in the present analysis, the Kaplan–Meier method was used to derive estimated rates of death and/or graft loss. Third, renal function was assessed at 1, 3, and 5 years after transplant using analysis of variance, linear mixed modeling and analysis of covariance, respectively 28, 29, 30. At 7 years after transplant, renal function was examined using a repeated‐measures model. The statistical approaches used evolved to reflect convention at the time of analysis. Compared with earlier time points, the methods used in the present report are more statistically robust. BODY.RESULTS: BODY.PATIENT DISPOSITION: In total, 543 patients composed the intent‐to‐treat population; of these, 128 of 184 belatacept MI–treated, 138 of 175 belatacept LI–treated and 108 of 184 CsA‐treated patients had data available for the analysis at month 84 (Figure 1). At month 84, 68.9% of all randomized and transplanted patients were assessed for death or graft loss (Figure 2). Notably, only a small number of patients declined to participate in the long‐term extension (belatacept MI, n = 3; belatacept LI, n = 0; CsA, n = 8). The median duration of follow‐up for patients randomized to belatacept MI, belatacept LI and CsA was 84.0 mo (range 0.03–84.0 mo), 84.0 mo (range 0.03–84.0 mo) and 70.8 mo (range 0.03–84.0 mo), respectively (Table S1). Figure 1 Patient disposition. Evaluable patients were defined as those who were followed for >84 mo or who had died or experienced graft loss by month 84. LI, less intense; MI, more intense. q2w, every 2 weeks; q4w, every 4 weeks. Figure 2 Proportion of patients assessed for death or graft loss. Data values are number (percentage). LI, less intense; MI, more intense. BODY.PATIENT AND GRAFT SURVIVAL: Kaplan–Meier estimated rates of death or graft loss for belatacept MI, belatacept LI and CsA were 19.0%, 17.1%, and 20.2%, respectively, at month 36; 26.5%, 25.6%, and 31.2%, respectively, at month 60; and 33.4%, 34.7%, and 35.5%, respectively, at month 84 (Table S2). At month 60, the HR for the comparison of belatacept MI with CsA was 0.874 (95% CI 0.583–1.310; p = 0.51), and the HR for the comparison of belatacept LI with CsA was 0.822 (95% CI 0.544–1.242; p = 0.35). At month 84, the HR for the comparison of belatacept MI with CsA was 0.915 (95% CI 0.625–1.339; p = 0.65), and the HR for the comparison of belatacept LI with CsA was 0.927 (95% CI 0.634–1.356; p = 0.70) (Figure 3A). Figure 3 Kaplan–Meier curve for (A) the composite end point of time to death or graft loss; (B) the individual contribution of death; (C) the individual contribution of death‐censored graft loss; and (D) the combined end point of first occurrence of death, graft loss or eGFR <20 mL/min per 1.73 m 2 . eGFR, estimated GFR; LI, less intense; MI, more intense. Kaplan–Meier estimated rates of death for belatacept MI, belatacept LI and CsA were 11.4%, 8.7%, and 9.5%, respectively, at month 36; 17.2%, 16.5%, and 18.5%, respectively, at month 60; and 24.9%, 26.7%, and 22.4%, respectively, at month 84 (Table S3). At month 60, the HR for the comparison of belatacept MI with CsA was 1.014 (95% CI 0.594–3B). Causes of death are summarized in Table S4. Kaplan–Meier estimated rates of death‐censored graft loss for belatacept MI, belatacept LI and CsA were 9.9%, 11.5%, and 12.8%, respectively, at month 36; 12.4%, 13.6%, and 18.0%, respectively, at month 60; and 12.4%, 13.6%, and 19.3%, respectively, at month 84 (Table S5). At month 60, the HR for the comparison of belatacept MI with CsA was 0.728 (95% CI 0.413–1.282; p = 0.27); the corresponding value for the comparison of belatacept LI with CsA was 0.815 (95% CI 0.469–1.415; p = 0.45). At month 84, the HR for the comparison of belatacept MI with CsA was 0.700 (95% CI 0.399–1.228; p = 0.21); the corresponding value for the comparison of belatacept LI with CsA was 0.783 (95% CI 0.452–1.353; p = 0.36) (Figure 3C). Causes of graft loss are summarized in Table S6. Kaplan–Meier estimated rates for the combined end point (first occurrence of death, graft loss or eGFR <20 mL/min per 1.73 m2) for belatacept MI, belatacept LI and CsA were 23.9%, 18.3%, and 30.0%, respectively, at month 36; 29.8%, 27.1%, and 40.2%, respectively, at month 60; and 37.4%, 36.8%, and 45.1%, respectively, at month 84. HRs comparing freedom from death, graft loss or eGFR <20 mL/min per 1.73 m2 from randomization to month 84 were 0.754 (95% CI 0.536–1.061; p = 0.10) for belatacept MI versus CsA and 0.706 (95% CI 0.499–0.998; p = 0.05) for belatacept LI versus CsA (Figure 3D). BODY.RENAL FUNCTION: From randomization to year 7, mean eGFR increased for both belatacept regimens but declined for CsA (Figure 4). Mean eGFR plus or minus standard error for belatacept MI at months 12, 36, 60, and 84 was 48.3 ± 1.3, 52.5 ± 1.4, 52.2 ± 1.7, and 53.9 ± 1.9 mL/min per 1.73 m2, respectively. The corresponding values for belatacept LI were 47.8 ± 1.3, 50.1 ± 1.4, 52.7 ± 1.6, and 54.2 ± 1.9 mL/min per 1.73 m2, whereas those for CsA were 40.3 ± 1.3, 38.0 ± 1.4, 35.8 ± 1.7, and 35.3 ± 2.0 mL/min per 1.73 m2. The estimated differences in GFR significantly favored each belatacept‐based regimen versus the CsA‐based regimen (p < 0.001 for overall treatment effect). Per the slope‐based model (and relative to month 1), patients randomized to belatacept MI or LI experienced a mean eGFR gain of 1.45 mL/min per 1.73 m2 (95% CI 0.94–1.96) and 1.51 mL/min per 1.73 m2 (95% CI 1.02–2.01) per year, respectively. Over the period from months 1 to 84, patients randomized to CsA had a mean decline in eGFR equivalent to −0.01 mL/min per 1.73 m2 (95% CI −0.55 to 0.52) per year. The GFR slopes diverged significantly between belatacept and CsA over time. The interaction of the treatment versus time effect deriving from the mixed‐effects model significantly favored each belatacept regimen versus CsA (each p < 0.001). Figure 4 Estimated mean GFR from months 1 to 84, as estimated via mixed‐effects modeling (without imputation). In this model, time was regarded as a categorical variable. For the sensitivity analysis in which GFR values that were missing due to patient death or graft loss were imputed as zero, mean eGFR plus or minus standard error for belatacept MI at months 12, 36, 60, and 84 was 43.9 ± 1.8, 42.5 ± 1.8, 39.0 ± 1.9, and 35.5 ± 2.0 mL/min per 1.73 m2, respectively. The corresponding values for belatacept LI were 44.1 ± 1.8, 42.5 ± 1.9, 39.6 ± 1.9, and 36.3 ± 1.9 mL/min per 1.73 m2. The corresponding values for CsA were 36.1 ± 1.8, 32.1 ± 1.9, 27.4 ± 2.0, and 25.5 ± 2.1 mL/min per 1.73 m2. With imputation, the effect of belatacept versus CsA at each time point remained statistically significant (p < 0.001 for overall treatment effect). Results from the slope‐based model with imputation showed that there was a mean decline in eGFR for all treatment regimens. The slope value was −0.77 mL/min per 1.73 m2 (95% CI −1.42 to −0.13) per year for belatacept MI and −0.80 mL/min per 1.73 m2 (95% CI −1.44 to −0.16) per year for belatacept LI. The corresponding value for the CsA‐based regimen was −1.17 mL/min per 1.73 m2 (95% CI −1.83 to −0.51). Compared with CsA, the slope estimates in the imputed analysis did not differ significantly for belatacept MI (p = 0.40) or LI (p = 0.43). BODY.ACUTE REJECTION: The Kaplan–Meier cumulative event rates of acute rejection for belatacept MI, belatacept LI and CsA were 19.3%, 18.6%, and 17.3%, respectively, at month 36; 21.1%, 19.5%, and 17.3%, respectively, at month 60; and 21.1%, 19.5%, and 17.3%, respectively, at month 84. At month 84, the HR for the comparison of belatacept MI with CsA was 1.22 (95% CI 0.75–2.00; p = 0.43); the corresponding value for the comparison of belatacept LI with CsA was 1.15 (95% CI 0.70–1.90; p = 0.59). Potential cases of suspected antibody‐mediated acute rejection were identified post hoc and are described in Table S7. BODY.DONOR‐SPECIFIC ANTIBODIES: The cumulative event rates of de novo DSAs at months 36, 60 and 84 for belatacept MI were 2.3%, 6.2% and 6.2%, respectively; the corresponding values for belatacept LI were 1.5%, 2.4%, and 4.5%, respectively, and the corresponding values for CsA were 11.3%, 17.1%, and 22.9%, respectively. At month 84, the HR for the comparison of belatacept MI with CsA was 0.26 (95% CI 0.11–0.59; p = 0.0001), and the HR for the comparison of belatacept LI with CsA was 0.18 (95% CI 0.07–0.46; p < 0.0001). Class I HLA specificity was detected in five belatacept MI–treated, three belatacept LI–treated, and 15 CsA‐treated patients. Class II HLA specificity was seen in two belatacept MI–treated and three CsA‐treated patients. In the CsA treatment arm, an additional four patients had DSAs with both class I and II HLA specificity. BODY.SAFETY: Serious AEs occurred in 87.0% of belatacept MI–treated, 89.1% of belatacept LI–treated and 84.2% of CsA‐treated patients. Infections were the most common serious AE. Incidence rates of serious infections per 100 person‐years of study drug exposure were similar across the treatment arms (Table 1). The incidence rates of any‐grade viral infections per 100 person‐years of treatment exposure were 20.98, 17.45 and 19.05 for belatacept MI, belatacept LI and CsA, respectively. The corresponding values for the incidence rates of any‐grade fungal infections per 100 person‐years of treatment exposure were 9.79, 6.93 and 11.00. The incidence rates of any‐grade malignancies per 100 person‐years of treatment exposure were similar across the treatment arms (Table 2). Table 1Cumulative incidence rates of selected serious adverse events adjusted per 100 person‐years of treatment exposure Belatacept MI (n = 184)Belatacept LI (n = 175)CsA (n = 184)Serious infectionsa , b 22.67 16.52 20.32 Urinary tract infection 3.02 3.62 3.54 Cytomegalovirus infection 2.20 1.94 1.71 Pneumonia 1.76 1.50 1.41 Pyelonephritis 1.44 0.69 1.83 Gastroenteritis 0.93 0.69 1.02 Herpes zoster 0.93 0.34 0.38 Sepsis 0.80 1.14 1.93 Urosepsis 0.68 0.80 0.76 Cellulitis 0.57 0.45 0 Gangrene 0.46 0.22 0 Pyelonephritis acute 0.46 0.11 0.91 Osteomyelitis 0.46 0.11 0.12 Bacteremia 0.34 0.11 0.63 Septic shock 0.23 0.22 0.75 Escherichia urinary tract infection 0.23 0.11 0.64 Bronchopneumonia 0.11 0.45 0 Serious gastrointestinal disordersc 6.3 6.2 6.8 Serious cardiac disordersc 5.2 4.1 5.2 Serious general disorders and administration site conditionsc 3.9 3.1 4.6 Serious blood and lymphatic system disordersc 3.5 2.4 2.5 Serious vascular disordersc 3.1 5.1 5.4 Serious investigations (laboratory parameters)c 2.0 2.2 4.4 Serious hepatobiliary disordersc 0.5 0.7 0.8 Serious endocrine disordersc 0.1 0.1 0.3 CsA, cyclosporine; LI, less intense; MI, more intense. aThe duration (patient‐years) of patient exposure to assigned study drug was calculated from the randomization date to the event date, to the date of last follow‐up or to month 84, whichever was earliest. bOnly preferred terms occurring in ≥2% of patients in any treatment arm are reported. cThe duration (patient‐years) of patient exposure to assigned study drug was calculated from the randomization date to the event date, to the date of last dose of study medication plus 56 days or to month 84, whichever was earliest. John Wiley & Sons, LtdTable 2Cumulative incidence rates of any‐grade malignancy adjusted per 100 person‐years of treatment exposure Belatacept MI (n = 184)Belatacept LI (n = 175)CsA (n = 184)Any malignancya , b 3.80 3.23 3.64 Basal cell carcinoma 1.05 0.69 1.55 Squamous cell carcinoma of the skin 0.93 0.68 0.51 Bowen's disease 0.46 0 0.25 Prostate cancer 0.23 0.46 0 CsA, cyclosporine; LI, less intense; MI, more intense. aThe duration (patient‐years) of patient exposure to assigned study drug was calculated from the randomization date to the event date, to the date of last follow‐up or to month 84, whichever was earliest. bOnly preferred terms occurring in two or more patients in any treatment arm are reported. John Wiley & Sons, LtdNine patients experienced posttransplant lymphoproliferative disorder (PTLD) prior to month 84 (n = 4, Epstein–Barr virus [EBV] positive; n = 5, EBV negative) (Table 3). Incidence rates per 100 person‐years of exposure in EBV‐positive patients treated with belatacept MI, belatacept LI and CsA were 0.12, 0.25 and 0.14, respectively. The corresponding values in EBV‐negative patients were 1.71, 5.19 and 0.00, respectively. Of those patients who had PTLD, five had primary central nervous system PTLD (n = 2, belatacept MI; n = 3, belatacept LI) and seven died (n = 3, belatacept MI; n = 4, belatacept LI). Two additional cases of PTLD were reported after month 84 but prior to database lock (n = 1, EBV‐positive patient randomized to belatacept MI; n = 1, EBV‐negative patient randomized to belatacept LI); the EBV‐negative patient randomized to belatacept LI who developed PTLD after month 84 also died. Table 3Cumulative incidence rates of PTLD adjusted per 100 person‐years of treatment exposure Time period, moPatients, n (incidence rate)Belatacept MI (n = 184)Belatacept LI (n = 175)CsA (n = 184)EBV positive 0–12 0 0 0 12–24 1 (0.63) 1 (0.68) 0 24–36 0 0 0 36–48 0 1 (0.93) 0 48–60 0 0 1 (1.37) 60–84 0 0 0 Overall 1 (0.12)a 2 (0.25) 1 (0.14) EBV negative 0–12 1 (7.89) 2 (11.88) 0 12–48 0 0 0 48–60 0 2 (25.28) 0 60–84 0 0 0 Overall 1 (1.71) 4 (5.19)b 0 CsA, cyclosporine; EBV, Epstein–Barr virus; LI, less intense; MI, more intense; PTLD, posttransplant lymphoproliferative disorder. aOne additional patient randomized to belatacept MI developed PTLD beyond month 84. bOne additional patient randomized to belatacept LI developed PTLD beyond month 84. John Wiley & Sons, Ltd BODY.DISCUSSION: In this analysis of the final 7‐year results from BENEFIT‐EXT, recipients of extended criteria donor kidneys randomized to belatacept had graft survival rates that were similar to those seen in patients randomized to CsA; however, fewer belatacept LI–treated than CsA‐treated patients met the combined end point of first occurrence of death, graft loss or eGFR <20 mL/min per 1.73 m2, which corresponded to a 29% reduction in risk (p = 0.05). An eGFR threshold of ≤20 mL/min per 1.73 m2 is clinically meaningful, given that patients with GFR values in this range are approaching end‐stage renal disease and the need for retransplantation. The improvement in GFR previously reported for belatacept versus CsA through 5 years of follow‐up was sustained and remained statistically significant at 7 years. The differences in eGFR between each belatacept‐based regimen and the CsA‐based regimen continued to increase over time. Trends from the slope‐based analysis with imputation are consistent with those from the slope‐based analysis without imputation; however, in the analysis with imputation, the difference between each belatacept‐based and the CsA‐based regimen did not remain statistically significant. A possible explanation is that there was clear differentiation of GFR slope estimates between regimens in the analysis without imputation, but by introducing zero values for GFR results that were missing due to death or graft loss, the slope estimates moved closer to each another; therefore, the difference between regimens became less pronounced, and statistical significance was lost. Notably, in a meta‐analysis of six clinical trials comparing belatacept with either CsA or tacrolimus, belatacept was associated with significantly greater eGFR at 12, 24 and 36 mo after transplant 32. This result is important from a clinical perspective because recipients of ECD kidneys tend to have statistically significantly lower GFR values than those who receive standard criteria donor kidneys 33, 34, 35, 36. A non‐nephrotoxic immunosuppressive regimen may help to maintain ECD renal function, delaying the time that recipients of such kidneys progress to chronic kidney disease, must return to maintenance dialysis and/or undergo retransplantation. As in earlier analyses of the intent‐to‐treat population of BENEFIT‐EXT 28, 29, rates of biopsy‐proven acute rejection were statistically similar across treatment arms. Most cases of acute rejection occurred prior to month 36; thereafter, one case of acute rejection was reported in each of the belatacept treatment arms. No patient randomized to CsA experienced acute rejection after month 36. Antibody‐mediated rejection has been identified as a major cause of late (>1 year after transplant) graft loss 21, 37, and the de novo development of DSAs is associated with significantly shorter graft survival 38, 39, 40, 41. In BENEFIT‐EXT, the cumulative incidence of de novo DSAs was statistically significantly lower in each belatacept‐based treatment arm versus the CsA‐based comparator regimen. This finding is consistent with data showing that the reduced strength of the CD28 signal, which is inhibited by belatacept, leads to a reduction in B cell responses 42; however, the significantly greater incidence of de novo DSA development among CsA‐treated patients also may have been the result of poorer treatment compliance. In BENEFIT‐EXT, information on study medication adherence was collected up to month 36 (Table S1). The incidence of serious AEs, serious infections, any‐grade viral or fungal infections, and any‐grade malignancies was similar across all treatment arms, suggesting that the safety profile of belatacept over the long term is similar to that of CsA. Nevertheless, the risk of PTLD was greater among belatacept‐ versus CsA‐treated patients, particularly those who were EBV negative prior to transplant. It is for this reason that belatacept is contraindicated in patients who are EBV negative or whose EBV serostatus is unknown prior to transplant 43, 44. In the present study, no difference in patient or graft survival was seen between belatacept‐ and CsA‐based immunosuppression. Analysis of the individual components of the composite end point showed an 11–12% increase in the risk of death and a 20–30% reduction in the risk of death‐censored graft loss for belatacept versus CsA at 7 years after transplant; neither sensitivity analysis was statistically significant. The lack of statistical significance for the composite end point in BENEFIT‐EXT contrasts with the survival advantage observed for belatacept versus CsA at 5 and 7 years after transplant in BENEFIT, which examined patients transplanted with a living or standard criteria donor kidney 45. In addition to kidney donor type, the discrepant findings between these two phase III trials can at least partially be attributed to the complementary rather than identical nature of the study populations: Kidney transplant recipients participating in BENEFIT were younger overall and had fewer comorbidities and thus were less likely to die with a functioning graft than were BENEFIT‐EXT participants. 46 or higher than 47 those reported in other randomized prospective registration trials. Moreover, almost 70% of the intent‐to‐treat population was evaluable for prospectively defined outcomes at month 84. Another limitation is that donor kidneys in BENEFIT‐EXT were protocol defined by extended criteria that incorporated but were not limited to those consistent with the former United Network for Organ Sharing (UNOS) definition of ECD kidneys 48. When the protocol for BENEFIT‐EXT was being developed in 2003, these kidney subtypes were considered high risk under UNOS and other published criteria available at the time 8, 48; however, definitions of high‐risk kidneys have since evolved. In 2013, the Kidney Donor Profile Index (KDPI) was introduced 49; therefore, the above findings are not necessarily predictive of outcomes that might be observed among recipients of donor kidneys with high (>85%) KDPI scores. This is important because not all ECD kidneys are equivalent in terms of anticipated patient outcomes; some ECD kidneys may have more favorable KDPI scores than others 50. Future studies should incorporate the newer KDPI scoring paradigm. In summary, in this 7‐year analysis of BENEFIT‐EXT, in comparison to CsA, belatacept was associated with similar rates of patient and graft survival and acute rejection, sustained improvements in renal function and significantly lower cumulative incidence of detectable de novo DSAs. BODY.DISCLOSURE: The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. A. Durrbach has received research support from Bristol‐Myers Squibb, and his spouse was once an employee of Bristol‐Myers Squibb. S. Florman and M. del Carmen Rial have received research/grant support from Bristol‐Myers Squibb. L. Rostaing has served on speakers' bureaus for Novartis, Astellas, Veloxis, Fresenius, and LFB. A. Matas has received grant support from Bristol‐Myers Squibb. T. Wekerle has received honoraria and research grants from Bristol‐Myers Squibb. M. Polinsky, H. U. Meier‐Kriesche, and S. Munier are salaried employees of Bristol‐Myers Squibb. J. M. Grinyó has served as an advisor to Bristol‐Myers Squibb, Chiesi, Veloxis, Quark, and Opsona. The other authors have no conflicts of interest to disclose. BODY.SUPPORTING INFORMATION: Data S1: Supplementary material. Table S1: Duration of follow‐up and treatment exposure in randomized, transplanted and treated patients. Table S2: Kaplan–Meier survival rate for time to death or graft loss from randomization to month 84 (year 7; all randomized and transplanted patients). Table S3: Kaplan–Meier survival rate for time to death from randomization to month 84 (year 7; all randomized and transplanted patients). Table S4: Causes of death. Table S5: Kaplan–Meier survival rate for time to death‐censored graft loss from randomization to month 84 (year 7; all randomized and transplanted patients). Table S6: Causes of graft loss. Table S7: Potential cases of suspected antibody‐mediated acute rejection. Click here for additional data file.

TITLE: The effect of adding oral oestradiol to progesterone as luteal phase support in ART cycles – a randomized controlled study ABSTRACT.INTRODUCTION: Luteal phase support in assisted reproductive technology (ART) cycles is still controversial. The present study was conducted to evaluate the effect of adding oral oestradiol to progesterone during ART cycles. ABSTRACT.MATERIAL AND METHODS: In this prospective case control study, infertile women under 35 years old who were candidates for IVF/ICSI cycles in Royan Institute were enrolled. A long gonadotropin-releasing hormone (GnRH) agonist protocol was used for ovarian stimulation. Patients were randomly divided into two groups for luteal phase support: the control group received vaginal administration of progesterone supplementation alone starting on the day after oocyte retrieval and continued until the tenth week if the chemical pregnancy test was positive. In the oestradiol group, 2 mg of oestradiol valerate was initiated orally with progesterone. The control group received a placebo instead of oestradiol. ABSTRACT.RESULTS: Ninety-eight women were studied as oestradiol (N = 47) and control groups (N= 51). There were no significant differences in the mean number of retrieved oocytes, number of transferred embryos, or chemical and clinical pregnancy rates between the two groups. Although the serum progesterone concentration was higher in the oestradiol group in comparison to the control group on day 7, 10 and 12 after embryo transfer, these differences were not statistically significant. ABSTRACT.CONCLUSIONS: The results suggested that adding oral oestradiol to vaginal progesterone supplementation does not improve the chemical and clinical pregnancy rates of IVF/ICSI cycles. BODY.INTRODUCTION: Several factors including oocyte quality and receptivity of endometrium influence the success of in vitro fertilization (IVF) cycles [1]. One of these influencing factors is implantation. Implantation of the embryo is critical in determining the success of assisted reproductive technology (ART) because after the transfer of high quality embryos, the pregnancy rate may still be relatively low [2]. Human embryo implantation is a three-stage process involving coordination between a receptive endometrium and a functional blastocyst [3]. Receptivity of the uterus is dependent on the hormonal status of the endometrium at the time of implantation [1]. Oestrogen causes proliferation of endometrial cells in the basal layer and increases progesterone receptors. Progesterone induces secretion from endometrial glandular cells and decidualization of the stromal layer. Therefore adequate concentrations of these hormones are essential for adequate endometrial maturation before embryo implantation [1]. Ovarian stimulation for IVF results in supraphysiological steroid levels and is associated with very low luteinizing hormone (LH) concentrations during the luteal phase [4]. Luteal phase supplementation (LPS), after controlled ovarian stimulation (COS) for IVF, has been a routine practice in IVF – embryo transfer (ET) because stimulated IVF cycles are associated with a defective luteal phase in almost all patients [5, 6]. Although it is generally agreed that LPS in IVF cycles improves the outcome, there is controversy over the best protocol [7]. There is a worldwide controversy concerning the type of hormones used for LPS, its dose and duration, and the time of starting and stopping it [8]. It is well established that luteal support with progesterone improves implantation in ART cycles [9]. Under progesterone supplementation, however, it has been shown that mid-luteal oestradiol (E2) levels decrease in a proportion of patients and that this might be associated with a concomitant decrease in pregnancy rates [10]. There are controversial reports about adding oestrogen to progesterone supplementation as LPS [1, 7, 11-13]. In the present study, the effect of adding oral oestradiol to progesterone for luteal phase support was compared to progesterone alone. BODY.MATERIAL AND METHODS: In this prospective case control study, infertile women under 35 years old who were candidates for IVF/intracytoplasmic sperm injection (ICSI) cycles in Royan Institute were enrolled. This study was approved by the Ethics Committee of Royan Institute and carried out between June 2005 and December 2007. Exclusion criteria consisted of age > 35 years, polycystic ovary syndrome (PCOs), high grade endometriosis, existence of myoma or adhesion in uterus, E2 level more than 3000 pg/ml on human chorionic gonadotropin (hCG) administration, and body mass index (BMI) more than 29 kg/m2 or less than 18 kg/m2. All patients signed an informed consent form. A long GnRH agonist protocol was used for ovarian stimulation. In this protocol, the women had first been downregulated with a GnRH analogue (Buserlin, Hoechst, Germany) which was administered 0.5 cc subcutaneously from the 21st day of the previous menstrual cycle. When pituitary suppression was achieved (on the second day of the menstrual cycle follicle-stimulating hormone (FSH) ≤ 5 IU/ml, LH ≤ 5 IU/ml, progesterone ≤ 1 ng/ml, oestradiol ≤ 50 pg/ml), its dose was reduced to 0.2 cc and 150-225 IU human menopausal gonadotropin (Menopur, Ferring, Germany) or 150-225 IU recombinant FSH (Gonal-F, Sereno, Italy) was administered intramuscularly from the 2nd day of the menstrual cycle daily. After 3 or more follicles had reached 17 mm in diameter, 10 000 IU human chorionic gonadotropin (Pregnyl, Daropakhsh, Iran) was used to induce oocyte maturation. Oocytes were aspirated transvaginally with ultrasound guidance 34-36 hours later. Then IVF or ICSI was done. Uterine embryo transfer was performed 48-72 hours after oocyte retrieval. Beta hCG was checked two weeks after embryo transfer. Clinical pregnancy was defined as the presence of at least one gestational sac with detectable fetal heart activity by transvaginal sonography. For luteal phase support, patients were randomly divided into two groups: group 1 (control) consisted of patients who received vaginal administration of progesterone supplementation 400 mg twice a day starting on the day after oocyte retrieval and continued until the tenth week if the chemical pregnancy test was positive. In the second group (oestradiol group), 2 mg of oestradiol valerate was initiated orally with progesterone. The control group received a placebo instead of oestradiol. In both groups, when the pregnancy test was negative or pregnancy loss occurred, the drugs used for luteal phase support were discontinued. For all hormonal evaluations, venous blood samples of all subjects were collected in Royan Institute’s Laboratory at the specified times. Blood samples were allowed to clot; sera were separated by centrifugation and examined immediately or stored at –20°C until assayed. The serum concentration of luteinizing hormone (LH, ELISA, Pishtaz-Teb, Tehran, Iran), oestradiol (E2, ELISA, DRG Instruments GmbH, Marburg, Germany) and progesterone (ELISA, DRG Instruments GmbH, Marburg, Germany) were checked on the hCG administration day and 7, 10 and 12 days after ET. The intra-assay and inter-assay variations (CV%, min-max) for LH, oestradiol and progesterone were 4.7-6.7% and 4.0-4.8%, 2.71-6.81% and 6.72-9.39%, 5.4-6.99% and 4.34-9.96%, respectively. BODY.STATISTICAL ANALYSIS: The program SAS 9.1 was used for data analysis. Results are presented as mean value ± standard error (SE). Repeated measure ANOVA, two sample t-test, Fisher’s exact test and χ2 test were used for the statistical analysis. P value less than 0.05 was considered significant. BODY.RESULTS: In this study, 98 women were selected and randomly studied as oestradiol (N = 47) and control groups (N = 51). The mean age of patients was 30.01 ±0.61 in the control group and 30.29 ±0.57 years in the oestradiol group (P > 0.05). There were no statistically significant differences between the two groups with respect to cause of infertility (Table I) and type of infertility (primary or secondary). The mean duration of infertility was 5.98 ±0.55 years in the control group and 7.81 ±1.37 years in the oestradiol group, but this difference was not statistically significant (P > 0.05). There were no significant differences between the two groups in LH levels measured on different days. Although the serum level of progesterone and oestradiol on different days in the oestradiol group was higher than that of the control group, these differences were not statistically significant (P > 0.05) (Table II). The mean number of retrieved oocytes, number of transferred embryos, and chemical and clinical pregnancy rate did not significantly differ between the two groups (Table III). In the oestradiol group, among women who had a positive β hCG test (25 out of 47), 23 women had an intrauterine gestational sac with detectable fetal heart while one woman had an ectopic pregnancy and in another woman a blighted ovum was detected. All women with positive chemical pregnancy in the control group (19 out of 51) had clinical pregnancy detected by sonography. BODY.DISCUSSION: Many areas of LPS used in ART cycles remain controversial. One of the subjects of debate in this regard is adding E2 to progesterone. In the present study, the use of oral oestradiol supplementation in combination with vaginal progesterone was evaluated and compared to progesterone alone during ART cycles where a long GnRH agonist protocol was used for controlled ovarian stimulation in all studied women. The results suggested that adding oral E2 did not improve chemical and clinical pregnancy rates. It is accepted that luteal function is suppressed in IVF cycles using GnRH agonists. This compromised function could potentially be attributed to ovarian stimulation and the resultant altered hormone levels, the process of oocyte retrieval and direct effect of the GnRH agonist on the corpus luteum [14]. Use of GnRH agonists may be associated with decreased production of E2 and progesterone in the luteal phase, a reduction in the length of the luteal phase, and impairment of endogenous gonadotropin secretion caused by persistent pituitary suppression [15]. Although there have been different clinical trials studying the effect of adding E2 to progesterone during LPS in ART cycles, these trials were different with regard to the type of oestradiol (oral, transdermal), its dose (2 mg or 6 mg), type of COS used (long or short GnRH agonist, GnRH antagonist) and type of progesterone used (intramuscular, vaginal). Some studies showed a positive effect of adding E2 in ART cycles. Farhi et al. studied the effect of adding E2 to progesterone supplementation during the luteal phase in IVF cycles with different GnRH agonist protocols (short or long). Their results showed that patients who received E2 supplementation and were treated with the long GnRH analogue protocol had significantly higher pregnancy and implantation rates [11]. In another study, Gorkemli et al. evaluated patients treated with a long ovulation induction protocol in two groups: group I used only 600 mg/day of progesterone vaginally and group II used 100 µg/day of transdermal oestradiol in addition to progesterone. They showed that adding oestradiol to progesterone in ICSI-ET cycles may increase implantation and pregnancy rates [12]. Other studies showed no benefits of adding E2. Engmann et al. evaluated the effect of E2 supplementation via the vaginal route on the overall probability of conception in subgroups of patients using the GnRH agonist suppression, GnRH antagonist, or microdose GnRH agonist protocol. Their findings suggested that the use of luteal phase vaginal E2 supplementation does not improve the overall clinical pregnancy rates but may increase the risk of biochemical miscarriage [1]. Serna et al. evaluated the effect of transdermal E2 administered after embryo transfer on cycle outcome. They found no difference between these luteal phase supports [7]. According to these different findings in different trials, in the present study, the serum progesterone and oestradiol levels were measured on different days after ET (7, 10 and 12 days) to compare between the two groups. The results showed that in spite of a mild increase in hormonal levels in the oestradiol group in comparison to the control group, these hormonal differences were not statistically significant. This may be one explanation for the similar pregnancy rate between these two groups in the present study. As we know, one phenomenon in COS is premature luteinization (PL), which is defined as a rise in serum progesterone levels on the day of hCG administration. An increase of absolute progesterone level on the day of hCG administration is considered as premature luteinization in most studies with different cut-off points (from 0.8 to 2 ng/ml) but some authors defined PL as a progesterone/oestradiol ratio of > 1 [16]. When we considered an increase of absolute progesterone level on the day of hCG administration as PL, this premature rise occurred in the oestradiol group but we did not find an obvious negative effect of it on pregnancy rate. One explanation for this finding is that the consequences of this premature elevation of serum progesterone on IVF outcome remain controversial [16]. Also it seems that using the level of a single sex hormone on the hCG administration day to predict pregnancy outcome in patients undergoing IVF is confounding [17]. In addition, we observed this PL in the group which was treated with oestradiol supplementation. It seems that oestradiol may affect the occurrence of PL. As Elnashar mentioned in his article, several mechanisms are considered to explain the PL, such as elevation of follicular LH levels, serum accumulation of hCG from HMG, increased LH receptor sensitivity of the granulosa cells to FSH or poor ovarian response with increased LH sensitivity [16], but none of them could completely explain this phenomenon. One of these mechanisms which may explain PL in the oestradiol group in the present study is increased LH receptor sensitivity of the granulosa cells to FSH, because it is suggested that the increased LH receptor sensitivity in the granulosa cells is due to higher cumulative exposure to oestradiol, which, in conjunction with FSH, could be one of the mechanisms to account for the premature increases in serum progesterone concentration [16]. Nevertheless, we could not draw any conclusion in this regard. Although adding oestradiol is a subject of debate in LPS, another controversy concerns the dose of oestradiol. Lukaszuk et al. in their prospective clinical trial evaluated the influence of different E2 supplementation doses (2 and 6 mg daily) during the luteal phase on implantation and pregnancy rates in women undergoing ICSI cycle. In their findings, significantly higher implantation rate and pregnancy rate were found in patients who received low dose E2 supplementation compared with no E2 supplementation, but the best outcomes were found significantly in the group with high dose E2 supplementation. They suggested that for women undergoing a long GnRH analogue protocol, addition of a high dose of E2 to daily progesterone supplementation significantly improves the IVF-ET outcomes [18]. Different doses of oestradiol were not compared in the present study and this may be one of its limitations. Another limitation of the present study is its relatively low sample size because it was necessary to take blood samples at least 5 times during the study and some patients did not consent to that. In conclusion, the present study showed that the addition of oral oestradiol with a dose of 2 mg daily to vaginal progesterone supplementation as luteal phase support did not improve chemical and clinical pregnancy rates of IVF/ICSI cycles. Further studies with a larger sample size and using different doses of oestradiol are recommended.

TITLE: Constraint-Induced Aphasia Therapy for Treatment of Chronic Post-Stroke Aphasia: A Randomized, Blinded, Controlled Pilot Trial ABSTRACT.BACKGROUND: Few studies have documented the possibility of treatment-induced improvements in language functions 12 months or longer after stroke. The purpose of the current study was to provide a preliminary estimate of efficacy of constraint-induced aphasia therapy (CIAT) when compared to no-intervention in patients with chronic (>1 year) post-stroke aphasia in order to provide the data needed to design an appropriately powered trial. ABSTRACT.MATERIAL/METHODS: This was a randomized, controlled, single-blinded, pilot trial. We identified 32 patients with chronic post-stroke aphasia. Of these, 27 were offered participation, and 24 were randomized (CONSORT diagram): 14 to CIAT and to 10 to no-intervention. CIAT groups received up to 4 hours/day of intervention for 10 consecutive business days (40 hours of therapy). Outcomes were assessed within 1 week of intervention and at 1 and 12 weeks after intervention and included several linguistic measures and a measure of overall subjective communication abilities (mini-Communicative Abilities Log (mini-CAL)). To maintain blinding, clinicians treating patients (CIAT group) did not communicate with other team members and the testing team members were blinded to treatment group assignment. ABSTRACT.RESULTS: Overall, the results of this pilot trial support the results of previous observational studies that CIAT may lead to improvements in linguistic abilities. At 12 weeks, the treatment group reported better subjective communication abilities (mini-CAL) than the no-intervention group (p=0.019). Other measures trended towards better performance in the CIAT group. ABSTRACT.CONCLUSIONS: In this randomized, controlled, and blinded pilot study, intensive language therapy (CIAT) led to an improvement in subjective language abilities. The effects demonstrated allow the design of a definitive trial of CIAT in patients with a variety of post-stroke aphasia types. In addition, our experiences have identified important considerations for designing subsequent trial(s) of CIAT or other interventions for post-stroke aphasia. BODY.BACKGROUND: Aphasia is one of the most devastating sequelae of stroke. It typically improves in the weeks and months after stroke, yet about 50% of patients are left with long-term residual deficits [1]. After the first year, spontaneous recovery is thought to be unlikely. Therapies administered during the first 12 months after stroke accelerate recovery [2,3]. Few studies have documented the possibility of treatment-induced improvements in post-stroke language functions after 12 months [4]. Traditionally, aphasia interventions utilize compensatory communication strategies to assist the participant in immediate communication needs. Multi-modal strategies include gesturing, writing, drawing, and augmentative low- and high-technology systems, with a common expectation that use of the alternative communication techniques will decrease naturally as the language capabilities increase. These interventions improve overall communication abilities, but questions have been asked whether they contribute to the recovery of language function, or whether they actually contribute to a learned non-use phenomenon [5]. To counteract the possibility of learned non-use, therapies utilizing restraint have been developed to mirror constraint-induced motor therapies. While it is relatively easy to restrain an unaffected extremity in motor therapies [6,7], restraining attempts to communicate non-verbally can be more difficult. Constraint-induced aphasia therapy (CIAT) encourages intensive verbal practice with supported verbal cuing while excluding the use of previously habitual compensatory strategies [8,9]. In CIAT, the theoretical model of Taub’s motor system for use-dependent cortical re-organization has been applied to a language-based program [8]. In this model, it has been postulated that the behavior of attempting to speak without success leads to communication frustration. This then results in fewer speaking attempts, more reliance on compensatory strategies, and less cortical stimulation in the language areas. The CIAT framework provides a structured supportive environment, with clinician guidance and shaping, positive reinforcement from group members, and social interaction opportunities. The theory is that the supportive environment and speaking opportunities will encourage more verbal attempts and stimulate cortical reorganization [10]. Thus, the goal of the present study was to provide evidence for the potential efficacy of CIAT when compared to no-intervention in patients with chronic (>1 year) post-stroke aphasia. More specifically, this randomized, controlled, blinded pilot study was conducted in order to estimate effect sizes, allowing the design of an appropriately powered trial [11]. BODY.MATERIAL AND METHODS: BODY.SUBJECTS: Subjects were recruited into this Institutional Review Board-approved study by word of mouth from among the stroke and aphasia clinics at the University of Cincinnati and University of Alabama at Birmingham, and from local aphasia support groups. We also listed the study on www.clinicaltrials.gov (registered NCT00843427; PI: Szaflarski), and several contacts were received directly from patients. After providing consent for screening, 32 individuals were interviewed. The inclusion criteria were chronic aphasia related to a single ischemic stroke in the left middle cerebral artery (LMCA) distribution (i.e., diagnosis of a single LMCA stroke was confirmed by medical record review including admission notes for the incident stroke and the results of brain imaging obtained prior to enrollment), Token Test in an impaired range (score ≤40), and pre-stroke fluency in English. The exclusion criteria were history of degenerative (e.g., dementia or Parkinson’s disease) or metabolic disorder (e.g., encephalopathy) or supervening illness (e.g., brain tumor or other cancer), history of severe depression or other mental illness, and positive pregnancy test in women of childbearing age. Patients with more than 1 stroke were not eligible. Five potential participants were excluded after interview. The format and the goals of the CIAT program were explained to all participants at the time of obtaining the informed consent. All patients indicated their understanding of the goals of the program prior to signing the informed consent; they also understood that they may be randomized to a no-intervention group, and that the follow-up testing will need to be performed. Of the 27 who were offered participation, 3 were excluded before randomization: 2 had a normal Token Test and 1 was hospitalized for reasons unrelated to the study. Fourteen subjects were randomized to receive CIAT (3–4 participants per group; 4 groups were assembled) and 10 to receive no-intervention (Figure 1). Demographic and clinical data of the participants are provided in Table 1. BODY.DEFINITIONS: After obtaining the informed consent, we initially screened the patients for the presence of aphasia with Token Test (TT) and categorized the severity of aphasia as mild (TT=40-37), moderate (TT=36-17) or severe (TT=16-0) [12]. Responders were defined as patients with at least a 20% relative improvement in at least 2 of the 5 primary scores in the 2-week period. Retainers were those patients whose 12-week score was not less than the 2-week score on at least 2 of the 5 outcome measures. BODY.DESCRIPTION OF THE INTERVENTION: The 2-week CIAT protocol was a closely monitored, individualized program embedded within a larger group activity [8,9]. To ensure consistency of the intervention, all clinicians completed a training program. Training (approximately 4–6 hours) was conducted by ALB prior to initiating any intervention session including basic theory of learned non-use, and the procedures for the treatment. Clinicians also viewed sample videos that exemplified the nature and set-up of the CIAT intervention, and they were taught a hierarchy of cues that ranged from most to least supportive (e.g., imitation to verbal reminder). During the first day of intervention, clinicians had no information on participants’ abilities as, by design, they did not participate in the initial evaluations. The measures were administered by coordinators who were blinded to group assignment (Figure 1). To enhance the chance for successful implementation of cues during the CIAT sessions, clinicians maintained a cuing tracking form that described the interaction between another clinician and their participant partners. The types of cues provided were tracked with a binary notation of whether the cuing resulted in a successful communication. This technique of behaviorally monitoring another clinician rather than self-monitoring was established in our previous study [9]. Clinicians and participants were rotated during each day’s session in order to provide a balance in communication partners. At the end of day 1, ALB studied the cuing tracking forms and the videos to create an individual treatment plan for the next day’s session. The treatment plan included identifying individual baseline linguistic strengths, suggested hierarchy of most beneficial cues, behaviors to constrain, and specific linguistic targets or goals [9]. The treatment plan was reviewed with the clinicians before the second day of therapy. From the second day onwards, clinicians promoted individualized support with cues at a linguistic level suitable for each person and continued data tracking. The tracking provided constant reminders to only provide cues that resulted in successful communication. Each day, clinicians reviewed the individual program and levels and made adjustments as needed. At the beginning of each session day, the clinicians reminded each participant of the goals and constraints that had been established for them. Goals followed a linguistic complexity hierarchy. For example, a participant who was strong in producing nouns, but limited in verbs, had a goal to add a verb to create a 2-word phrase. A linguistic complexity chart was provided to the clinicians to support the progression. If a participant demonstrated a milder aphasia, then the D-level hierarchy of sentences was implemented [13]. The treatment program for each of the 3–4 participant groups lasted 2 weeks, with direct therapy for about 4 hours per day for 10 consecutive weekdays. The sessions were organized into 4 45-minute periods, with a 10–15 minute break in between each session. Socialization between clients and clinicians was encouraged throughout the program, even during the break periods. At the beginning of each session, participants were dealt cards and they were instructed to play a go-fish type game. The cards provided the opportunity for the participants to interact with each other during an ongoing game that engaged participants’ visual, attention, and memory skills, although these were not specifically targeted. The cards provided visual stimuli of line drawings of nouns (singular, plural to elicit numbers, and with colors) and photos of action verbs [8,9]. The program included feedback given to the clinician, with guidance provided by ALB. Clinicians were encouraged to use redirecting phrases such as “try again” and “are you sure?” rather than using negative responses. If the other participants responded positively and the communicative exchange was successful, clinicians were instructed not to correct; communicative success is more important than the sentence accuracy. Whenever clinicians were observed to manage cards for the participant, this was discouraged; clinicians were trained to be supportive while encouraging independence. Consistent with the current standards of care, the no-intervention group did not receive any specific treatment and the participants were asked to continue all previous activities as usual. All participants were asked not to take part in any other intervention during their involvement in the study and all complied. BODY.RANDOMIZATION: Patients were randomized by the study statistician (CJL) after the patients received all pre-requisite activities (consenting, clinical record review, neuropsychological aphasia testing (NAT)). Patients were assigned to receive either 2 weeks of CIAT, or no-intervention, and then to undergo NAT within one week and 3 months of CIAT completion (Figure 2 – CONSORT Diagram). We used a simple scheme that randomized each block of patients to either CIAT or control. Randomization occurred after consent, and with the statistician blinded to participant performance on screening and baseline testing. We did not replace subjects who do not complete the full 2 weeks of therapy. BODY.BLINDING: After randomization, sealed study charts containing all pre-intervention testing results (NAT) were funneled through the study biostatistician (CJL) to the therapists. Therapists set up the intervention groups so that the coordinators (CB, ANM) who collected all NAT data throughout the study remained blinded to group assignment. Participants were asked not to reveal their group assignment to coordinators during the post-treatment interactions. BODY.MEASURES: The Token Test was used only for primary screening and study qualification. All participants received NAT which included: (1) the Boston Naming Test (BNT) [14], (2) the Controlled Oral Word Association Test [15], (3) the Semantic Fluency Test (SFT) [15,16], (4) the Complex Ideation subtest from the Boston Diagnostic Aphasia Examination (BDAE) [17], (5) the Peabody Picture Vocabulary Test III (PPVT III) [18], and (6) the Mini-Communicative Activities Log (Mini-CAL) which is a subjective measure of communicative abilities [8,9]. The study coordinators, who were extensively trained in the use of these measures and blinded to group assignment, administered the NAT measures within 1 week prior to CIAT and again during both the first week the twelfth week following CIAT completion. All data were entered into REDCap (Research Electronic Data Capture) for subsequent analysis [19]. BODY.DATA ANALYSIS: Analysis was conducted on an intent-to-treat basis. One patient did not complete the 12 week visit or testing, and 7 additional patients were missing at least 1 NAT score. Missing scores were left missing, and are excluded from analysis. The primary analysis used Independent Samples T-Tests to examine the differences in NAT scores at each time point between the intervention and control groups, and effect sizes and 95% confidence intervals were calculated. The secondary analysis was an ad hoc analysis comparing the characteristics of patients who demonstrated a response or change in NAT scores to patients who did not. Independent T-Tests, The Mann-Whitney U test, and Fisher’s Exact Test were used to compare patient characteristics between patients who demonstrated a response or change and patients who did not. All statistical analyses were conducted using SPSS 22.0 (IBM Corporation, Armonk, NY) and R 2.15.3 (base package). BODY.RESULTS: BODY.SUBJECTS: There were 24 patients enrolled in the study, 14 in the intervention group, and 10 in the control group (see CONSORT Diagram). Two patients in the intervention group did not complete the study, 1 due to lack of transportation and 1 due to hospitalization with an illness unrelated to the study. Overall, there were no significant differences in demographic characteristics and past medical history between the 2 groups (Table 1). The mean age was 57 (SD±11) years in the intervention group and 51 (SD±13) years in the control group. Most patients were Caucasian, 10/14 (71%) in the intervention group and 9/10 (90%) in the control group. BODY.PRIMARY ANALYSIS: There were few statistically significant differences in NAT scores between the intervention and control groups (Table 2). Specifically, differences were observed in subjective communication abilities assessed with the mini-CAL. Patients who received CIAT scored higher on the mini-CAL at 12 weeks after intervention compared to the control group (Mean 31 vs. 23; difference=8, 95% CI 1.3 to 13.5, p=0.019). The SFT was marginally higher 2 weeks after intervention in CIAT patients than controls (Mean 21 vs. 12; difference 9, 95% CI −0.3 to 17.8, p=0.058). This difference was not sustained at twelve weeks after intervention. Scores for all NATs are presented in Table 2 and Figure 3. BODY.SECONDARY ANALYSIS: Overall, 5/24 (21%) of participants could be classified as a “responders”, as defined in the methods section. Of these, 4/5 (80%) were in the CIAT group. Complete characteristics of responders and non-responders are presented in Table 3. Responders were less likely to have a history of hypertension 0/5 (0%), compared to non-responders 10/14 (53%), p=0.053. Of the 5 patients who demonstrated a change in NAT scores during the course of the study, only 1 (20%) retained the change at the twelve-week visit. BODY.DISCUSSION: In this prospective, preliminary, randomized, blinded study of patients with chronic post-stroke aphasia, we estimated the effect of CIAT on linguistic performance. While the results are largely not statistically significant, a few points need to be discussed. First, the study included patients with highly variable levels of post-stroke aphasia, making the comparisons between the groups somewhat difficult. Second, the observed effects are consistent with improvement in the CIAT group beyond that in the control group, with trends towards statistical significance for some of the variables even in this small sample. Defining a responder as a participant with 20% improvement on at least 2 of the 5 tests, then the number needed to show significant between-group differences would be 62 and allows for the design of a more comprehensive trial. Finally, a significant improvement was noted in the mini-CAL results for the CIAT group compared to controls. This indicates that, at least subjectively, the patients in CIAT group perceived more improvement over time than participants in the control group. Reasons for the participants’ perception of improvement, as measured by the mini-CAL, may be related to the social activity of the therapy sessions. In order to isolate the variable of social interaction from CIAT theory, future studies should consider including a study arm involving exposure to social interaction with the same duration and frequency as the CIAT group, but without the CIAT intervention. This could also assess for possible placebo effects [20]. A preliminary qualitative investigation suggested a relationship with social factors [21]. Social factors may be related to both clinicians and other participants in the group. To date, few reports on the potential efficacy of CIAT have been published. Five studies in patients with chronic aphasia have included both a treatment group and a control group [8,22–25]. Only in the original study were participants randomized to treatment group [8]; however, unlike the current study, the personnel collecting linguistic data were not blinded to treatment assignment in any of these studies. In the original study, treatment intensity was much higher in the CIAT group [8]. In the other 4 studies CIAT was compared to other treatment approaches – either model-based aphasia therapy (training based on functional deficit) [25], CIAT plus written module [24], PACE [23], or model-oriented aphasia therapy (MOAT) [22]. In addition to the above studies, a recent study compared modified CIAT to conventional aphasia therapy in patients with less than 4 months (subacute) since the incident ischemic or hemorrhagic stroke [26]. All these studies showed that while participants receiving treatment improved linguistic performance, between-group differences were minimal, if any. Thus, when comparing the results of the original study by Pulvermuller et al. and the results of the subsequent studies, including ours, the questions of training intensity and social interactions between participants, and participants and clinicians need to be considered; both can contribute to improved communicative abilities via increased verbal communication practice [8,21,24]. This demonstrates the importance of comparing treatment approaches against a non-active control. We did not show any statistically significant improvement in objective tests of linguistic performance from before to after treatment. One of the reasons for this lack of statistical significance in our small sample size is the high variability in language scores observed in both groups (Figure 3 We identified important considerations for study design that will minimize unnecessary variability and maximize the potential for observing treatment benefit. Treatment fidelity needs to be monitored, either via independent reviewers performing video or by direct review of the conducted sessions. A transfer package similar to that offered in motor rehabilitation studies will need to be developed and applied to this group [27]. Also, the outcome measures should include measures of pre- and post-intervention discourse and assess changes in the severity of aphasia using, e.g., the Western Aphasia Battery-Revised [28]; assessing linguistic complexity using mean length of utterances [29]; or indices of syntactic form [30]. In addition to the above, stratification by severity, education, and socioeconomic status might emphasize the magnitude of improvement relative to the degree of aphasia [31]. Finally, other factors, including the effects of lesion size and location on changes in aphasia diagnosis (type) during the therapy session, should be assessed [32,33]. BODY.CONCLUSIONS: Modest improvements were noted in the CIAT group on the collected objective tests that were not noted in the control group, forming the basis for estimating effect sizes needed for a comprehensive study. Moreover, the CIAT group reported significant subjective improvement that confirms the importance of this approach. Larger randomized controlled trials that adopt the measures suggested to reduce variability and maximize observable benefit are warranted.

TITLE: Effects of Arabinoxylan and Resistant Starch on Intestinal Microbiota and Short-Chain Fatty Acids in Subjects with Metabolic Syndrome: A Randomised Crossover StudyArabinoxylan, Resistant Starch and SCFA Recently, the intestinal microbiota has been emphasised as an important contributor to the development of metabolic syndrome. Dietary fibre may exert beneficial effects through modulation of the intestinal microbiota and metabolic end products. We investigated the effects of a diet enriched with two different dietary fibres, arabinoxylan and resistant starch type 2, on the gut microbiome and faecal short-chain fatty acids. Nineteen adults with metabolic syndrome completed this randomised crossover study with two 4-week interventions of a diet enriched with arabinoxylan and resistant starch and a low-fibre Western-style diet. Faecal samples were collected before and at the end of the interventions for fermentative end-product analysis and 16S ribosomal RNA bacterial gene amplification for identification of bacterial taxa. Faecal carbohydrate residues were used to verify compliance. The diet enriched with arabinoxylan and resistant starch resulted in significant reductions in the total species diversity of the faecal-associated intestinal microbiota but also increased the heterogeneity of bacterial communities both between and within subjects. The proportion of Bifidobacterium was increased by arabinoxylan and resistant starch consumption (P<0.001), whereas the proportions of certain bacterial genera associated with dysbiotic intestinal communities were reduced. Furthermore, the total short-chain fatty acids (P<0.01), acetate (P<0.01) and butyrate concentrations (P<0.01) were higher by the end of the diet enriched with arabinoxylan and resistant starch compared with those resulting from the Western-style diet. The concentrations of isobutyrate (P = 0.05) and isovalerate (P = 0.03) decreased in response to the arabinoxylan and resistant starch enriched diet, indicating reduced protein fermentation. In conclusion, arabinoxylan and resistant starch intake changes the microbiome and short-chain fatty acid compositions, with potential beneficial effects on colonic health and metabolic syndrome. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT01618526 BODY.INTRODUCTION: Increased consumption of dietary fibre (DF) has been shown to improve components of metabolic syndrome (MetS), including dyslipidaemia, insulin sensitivity and abdominal obesity,[1–3] which are well-known risk factors for cardiovascular disease and type 2 diabetes. These beneficial effects are most likely modulated through the actions of DF on certain bacteria in the colon[4] and the capabilities of these bacteria to produce an array of small organic molecules, including short-chain fatty acids (SCFAs).[5] Increased production of SCFAs in general and of butyrate in particular may protect against diet-induced obesity and improve insulin sensitivity.[6–8] SCFA production depends on the type of ingested DF, the microbiota, the colonic transit time and the colonic pH.[9] Resistant starch (RS) is a homo-polysaccharide of glucose[10] resistant to digestion in the upper gastro-intestinal tract[11] because of physical indigestibility (type 1), granularity (type 2), retrogradation during heating (type 3), or chemical modification (type 4).[10] RS type 2 is a readily available pure form of RS that can be obtained in sufficient quantities for human studies. Previously, RS type 2 has been shown to augment butyrate production[12, 13] and to modify the composition of the intestinal microbiota[5, 14–16] typically by increasing the proportions of Bifidobacterium species, Ruminococcus bromii[14, 17] and Eubacterium rectale.[14] Studies on RS type 3 have also been conducted and shown enhancement of Ruminococcus bromii and Eubacterium rectale,[18, 19] whereas RS type 4 apparently demonstrate different functional effects on the microbiota by augmentation of Bifidobacterium adolescentis and Parabacteroides distasonis.[14] Arabinoxylan (AX) is a hetero-polysaccharide consisting of a linear xylose backbone with variable arabinose substitution and cross-linking with ferulic acid, depending on the grain species and fractions.[20] It is the main DF component in wheat and rye[20] and has also been shown to enhance SCFA and butyrate production.[3, 21] The proportions of Bacteroides, Prevotella, Roseburia and Bifidobacterium species have been shown to be increased after an AX-enriched high-fat diet in mice,[22] whereas AX provided in a whole-grain rye diet in a human intervention study failed to show changes in the microbiota.[23] Both RS and AX have been found to improve metabolic parameters in humans,[24–26] but there is little knowledge of how these fibres in combination influence the microbiome and SCFA production in prediabetic subjects, i.e. those with MetS. The objectives of the current crossover study of subjects with MetS were to determine how an AX- and RS-enriched diet, hereafter referred to as a healthy-carbohydrate diet (HCD), alters the microbiome and SCFA concentrations in faeces compared with a low-fibre Western-style diet (WSD). We hypothesised that AX and RS would beneficially change the intestinal microbiota and enhance colonic SCFA production in general and butyrate in particular compared with the WSD. BODY.MATERIALS AND METHODS: BODY.STUDY DESIGN AND SUBJECTS: The dietary intervention study was conducted according to the guidelines in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Central Denmark Region Committees on Biomedical Research Ethics (journal no. 1-10-72-122-12) March 29 2012 (S1 Information). Written informed consent was obtained from all subjects prior to screening. The study was registered at ClinicalTrials.gov ID: NCT01618526 (May 30 2012) and NCT01584427 (April 20 2012). The delay between approval and registration was due to maternity leave for one of the study coordinators. We confirm that all ongoing and related trials for this intervention are registered. The study was performed as a randomised crossover, open-label study with two 4-week intervention periods and an intermediate washout period of a minimum of four weeks. Recruitment took place via advertisements in local newspapers from May 9 2012 to November 30 2012. The study was carried out at Aarhus University Hospital, Aarhus, Denmark between June 2 2012 and April 11 2013. Twenty-two subjects between 39 and 75 years of age with MetS[27] were included in the study. Two more subjects were included than original described in the trial protocol. We excluded subjects if they had a history of diabetes or gastrointestinal disease or hepatic, renal, cardiovascular or uncontrolled metabolic disease. Alcohol abuse, the daily use of non-steroidal anti-inflammatory drugs, pregnancy and lactation were additional exclusion criteria. BODY.INTERVENTION DIETS: The HCD was formulated with a high concentration of DF based on AX- and RS-enriched cereal foods, whereas the WSD was based on refined grains and a minimal concentration of DF. AX was obtained from whole-grain rye and enzyme-treated wheat bran, and RS was provided as RS type 2[28] from raw potato starch and high-amylose maize starch. The diet allocation sequence was determined by the study coordinators using a simple randomisation method.[29] The cereal key foods were incorporated into the subjects’ habitual diets (see dietary counselling) and constituted approximately 50% of their total daily energy needs. The diets were isocaloric, as estimated by nutrition labelling. The HCD included experimentally prepared bread rolls (combo rolls) and pancakes (combo pancakes) (Lantmännen R&D, Stockholm, Sweden) that contained RS (7.0 g per roll and 8.4 g per pancake) in the form of high-amylose maize starch (HI-MAIZE260®) (Ingredion Incorporated Inc., Bridgewater, NJ, USA) and AX (6.0 g per roll and 8.4 g per pancake) in form of enzyme-treated wheat bran[30] (DuPont Nutrition and Biosciences ApS, Brabrand, Denmark). RS in the form of raw potato starch (24 g per day) was provided by KMC (Brande, Denmark). Raw potato starch was consumed unheated and dissolved in a smoothie or water. Rye flakes (Lantmännen Cerealia A/S, Vejle, Denmark), rye bread (Lantmännen Schulstad A/S, Hvidovre, Denmark), rye pasta (Il Fornaio, Corte Madera, California, USA) and smoothies (Rynkeby, Ringe, Denmark) were commercially available products. For the WSD, the subjects received corn flakes and wheat bread (Coop Denmark A/S, Albertslund, Denmark), spelt bread rolls (Lantmännen Unibake, Horsens, Denmark), wheat pasta and pancakes (Lantmännen Cerealia A/S, Vejle, Denmark), and smoothies (Rynkeby, Ringe, Denmark). The key foods were provided at two-week intervals. Due to the different appearances of low and high-fibre key foods, single-blinding of the participants, as described in the original trial protocol, was not feasible. The nutritional compositions of the key foods are shown in Table 1. 10.1371/journal.pone.0159223.t001Table 1BODY.DAILY INTAKE OF NUTRITIONAL CONSTITUENTS OF THE KEY FOODS IN THE WESTERN-STYLE DIET (WSD) AND THE HEALTHY-CARBOHYDRATE DIET (HCD).: WSDHCDEnergy (KJ)a 5280 4722 Protein (g) (E%) 40.4 (13) 31.7 (11) Fat (g) (E%) 17.3 (12) 17.3 (14) Digestible carbohydrates (g) (E%) 225.6 (73) 180.8 (65) Sugars (glucose, fructose, sucrose) (g) 27.0 25.9 Lactose (g) 2.7 2.4 Digestible starch (g) 195.9 152.5 Non-digestible carbohydrates (g) 14.2 59.0 Resistant starch (g) 2.8 20.7 Non-starch polysaccharides (g) 8.5 32.3 Cellulose (g) 1.2 4.2 Arabinoxylan (g) 3.6 16.0 LMW non-digestible carbohydrates (g) 2.9 6.0 Fructan (g) 2.1 5.3 Arabinoxylan oligosaccharides (g) 0.8 0.7 Lignin (g) 3.4 5.0 Dietary fibre (g)b(E%) 17.6 (3) 64.0 (11) E%, energy percentage; LMW, low molecular weight. aCalculated as the sum of: 17 kJ per g digestible carbohydrate 8 kJ per g dietary fibre, 17 kJ per g protein and 37 kJ per g fat. bCalculated as the sum of non-digestible carbohydrates and lignin. BODY.DIETARY COUNSELLING, ENERGY INTAKE AND COMPLIANCE: A clinical dietician instructed each subject at the beginning of both interventions. Based on individual energy need, a dietary plan was developed to maintain stable body weight and to limit DF except for that obtained from the key foods. The subjects were requested to maintain their regular lifestyles, including physical activity, smoking habits, alcohol intake and medication, throughout the study. The subjects were provided with electronic kitchen scales and checklists of the key food items to ensure dietary adherence. Before and during the diets, the subjects completed food records on three consecutive days, one of which was a weekend day. The macronutrient compositions of the habitual diets were calculated by Master Dietist System version 1.235 (2007) based on the Danish National Food Administration Database. The macronutrient compositions of the intervention diets were calculated as the sum of the compositions of the key foods and what the subjects ingested in addition to the key foods according to the food records. We assumed that the subjects consumed all of the key foods delivered. However, data were missing from seven food records (3 pre-diets, 2 HCD and 2 WSD) due to insufficient registration. BODY.RECORDING OF GASTROINTESTINAL SYMPTOMS AND STOOL PARAMETERS: An internally validated questionnaire based on verified scoring systems and rating scales[31–34] was used to assess general wellbeing and gastrointestinal symptoms. Five gastrointestinal symptoms (abdominal pain, bloating, rumbling, flatulence and nausea) and their severities as well as general wellbeing and health concerns were scored on visual analogue scales. We also assessed stool parameters, such as bowel movements, consistency, urge and occurrence of mucus. The subjects were instructed to fill in the questionnaires before and after each dietary intervention. In addition, they registered the number and consistency of stools on the same three days as they completed the food records. BODY.FAECAL SAMPLING AND HANDLING: Faecal samples were collected using EasySampler® Stool Sample Collection (Alpha Laboratories Ltd, Hampshire, UK) before and at the end of each dietary intervention. The samples were immediately stored at -20°C, and within 24 hours, they were moved to storage at -80°C without being thawed. BODY.BODY WEIGHT AND FAT PERCENTAGE: Measurements of waist circumference, body weight (TANITA WB-100A CLASS 111), and body fat percentage (Body Fat Monitor F306, OMRON, Hoofddorp, The Netherlands) were assessed before and at the end of each intervention when the subjects were fasting, wearing one layer of light clothes and had just emptied their bladder. BODY.CHEMICAL ANALYSES: The total RS levels in foods were determined by the AOAC method (AOAC Official Method 2002.02), as described by McCleary & Monaghan[35] and non-starch polysaccharides (NSPs) in foods and faeces, as described by Bach Knudsen[36] except that acid hydrolysis was performed in 2 M H2SO4 for one hour instead of in 1 M H2SO4 for two hours. When analysing the two RS sources, raw potato starch and HI-MAIZE260®, with the NSP procedure, we discovered that a fraction of starch in HI-MAIZE260® withstood gelatinization and hydrolysis and was analysed as part of the NSP fraction. To avoid this interference, dimethyl sulphoxide (DMSO) was used to disperse the RS[37] in the NSP procedure. The RS levels estimated with the two methods were designated as RSENZ and RSDMSO, respectively. The other analytical methods used to characterise the chemical compositions of the food items have been previously described by Nielsen et al.[15] The concentrations of faecal SCFAs and other acids including lactic acid were determined by gas-liquid chromatography (HP-6890 Series Hewlett Packard Palo Alto, CA) according to Jensen et al.[38 BODY.BACTERIAL GENOMIC DNA EXTRACTION AND 16S RRNA GENE SEQUENCING: Genomic DNA was extracted in duplicate from four faecal samples per subject (before and after WSD and HCD consumption) using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). Approximately 200 mg was chipped from each sample without thawing and placed directly into sterile 2 ml tubes containing 16 ml QIAamp InhibitEx buffer and 300 mg of 0.1 mm diameter zirconia/silica beads (Biospec Products, Bartlesville, Oklahoma, USA). The samples were shaken twice for 1 min each time at 6.5 m/s in a MP FastPrep-24 tissue and cell homogeniser (MP Bio, Santa Ana, California, USA) prior to completing the DNA extractions and purifications, according to the manufacturer’s instructions for pathogen detection. DNA concentration was measured using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA) and diluted to 20 ng/μL. Equal volumes of duplicate DNA extractions from each faecal sample were combined prior to PCR amplification. The 16S rRNA V4 region was amplified by PCR using the barcoded F515 forward primer and R806.[39, 40] Amplification was performed using Ex Taq DNA Polymerase (TaKaRa, Otsu, Japan) for 30 cycles of 94°C for 45 seconds, 54°C for 60 seconds, and 72°C for 30 seconds. PCR products were purified using a Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, Wisconsin, USA), and the pooled amplicons were sequenced with an Illumina MiSeq at the Genome Center DNA Technologies Core, University of California, Davis, California, USA. Raw Illumina FASTQ sequences were analysed using Quantitative Insights Into Microbial Ecology (QIIME) software package version 1.8.0.[41] Demultiplexing and quality filtering were performed with the default settings, except that a minimum average quality score of 30 was used for quality filtering instead of the default score of 25, and the reverse primer sequences were removed. The open-reference OTU picking strategy in QIIME was used to select operational taxonomic units (OTUs) with 97% sequence identity to sequences in Greengenes (release 13_8) database[42] and to each other, according to the UCLUST algorithm.[43] OTUs similar to chloroplasts or those found in very low abundance (< 0.005%)[44] were removed from the OTU table prior to further analysis. The Chao1 diversity index, Phylogenetic Diversity Whole Tree,[45] and observed species were determined for increasing numbers of randomly sampled sequences per sample to generate alpha diversity rarefaction curves. The curves became asymptotic at 12,000 sequences per sample and therefore, this number of randomly sampled DNA sequences was used for calculation of the weighted UniFrac metric[46] in QIIME. BODY.STATISTICAL METHODS: Prior to conducting the study, a sample size was calculated based on an expected increase in the faecal butyrate concentration after HCD consumption compared with that after WSD consumption. Based on a Danish study population treated with approximately 18 g of dietary fibre (Plantago ovata seeds) for 4 weeks,[47] we anticipated that the standard deviation of the mean difference in butyrate concentrations between the diets would be 7.5 mmol/l and that the minimal relevant difference between means would be 7.5 mmol/l–thus we used a standardized minimal relevant difference of 1 in a crossover design. The power calculation was done using software developed by Schoenfeld[48] and by formulas given by Julious et al.[49] The total number of subjects needed to obtain a statistical power of 90% for a type I error of 5% in a crossover trial was calculated to be 13. The anticipated dropout rate was set to 33%. Descriptive statistics are given as the median with the interquartile range (IQ). Analysis of variance for repeated measurements was performed to examine faecal SCFA and carbohydrate residue responses using subject, diet, period, and baseline values as covariates. These data were log-transformed to obtain a Gaussian distribution. Model validation was performed by inspection of Bland-Altman plots to check if the mean and standard deviation of the differences were constant throughout the range[50] and by probability plots of the residuals. Additional statistical analyses of the total SCFA, acetate, propionate and butyrate concentrations were carried out, excluding the two participants receiving the anthelmintic treatment, which had no effect on the results. The dietary data, body measurements, data from the questionnaires and differences from the baselines to the end of the diets were compared with the two-tailed paired t-test. Model validation was performed by Bland-Altman plots and probability plots of the differences. Wilcoxon signed-rank tests were performed for comparison of the DF intakes, protein intakes, faecal characteristics and gastrointestinal symptoms, because the assumptions for the two-tailed t-test were not feasible. These statistical analyses were conducted using STATA/IC 12.1 (StataCorp, College Station, Texas, USA), and Graph Pad Prism 6 (Graph Pad Software, Inc., La Jolla, California, USA) was used to generate graphical elements. Differences in UniFrac distances between samples from different experimental groups were tested by the Wilcoxon signed rank test followed by Bonferroni correction of the resulting p-values. Bacterial taxa that were present at greater than 0.1% abundance in at least 20% of the samples from 18 subjects who collected faecal samples throughout the entire study period were evaluated using R[51] with the paired Wilcoxon signed-rank test followed by Benjamini and Hochberg false discovery rate adjustment of the p-values. Alpha diversity measurements were compared in the same manner as the bacterial taxa comparisons. Differences were regarded as significant at a P < 0.05. BODY.RESULTS: BODY.STUDY POPULATION: A total of 67 adults were screened and 22 met the inclusion criteria and were randomized between June 2012 and January 2013 (Fig 1) (S2 Information). Nineteen subjects completed the study. Two subjects withdrew for reasons unrelated to the study, and one left the study during the first week of the HCD because of abdominal discomfort. Seven subjects were treated with antihypertensive drugs, and seven were treated with cholesterol-lowering medication at consistent doses throughout the study. Two subjects received treatment with mebendazole for 4 weeks (HCD) due to pinworm infection. The baseline characteristics and habitual macronutrient intakes of the subjects are shown in Table 2. 10.1371/journal.pone.0159223.g001Fig 1BODY.SUBJECT FLOW.: 10.1371/journal.pone.0159223.t002Table 2BODY.BASELINE CHARACTERISTICS AND HABITUAL MACRONUTRIENT INTAKES.: MedianInterquartile rangeTotal rangeAge (years) 60 48–67 39–75 BMI (kg/m2) 30.6 29.3–35.2 25.9–41.0 Waist (cm) 106 103–116 89–130 Body fat (%) 33 30–39 25–49 Energy intake (KJ/day)a 7812 6920–8845 4600–13991 Protein (g)a 79 69–91 53–140 Fat (g)a 70 56–94 32–160 Total carbohydrates (g)a 214 172–234 130–370 Digestible carbohydrates (g)a 197 154–210 117–335 Dietary fibre (g)a 18 14–25 8–35 aBased on dietary records from first run-in (n = 19). BODY.DIETS, ENERGY INTAKE AND ANTHROPOMETRIC DATA: There were no significant differences in the energy intake (P = 0.75) of the subjects or their consumption of protein (P = 0.91), fat (P = 0.82), carbohydrates (P = 0.36) or DF (P = 0.12) during the two run-in periods. The baseline characteristics and habitual macronutrient intake from first run-in period is shown in Table 2. The DF concentration in the key foods was 64 g/day for the HCD and 18 g/day for the WSD (Table 1). Thus, the subjects increased their total DF intake from a median of 18 g/day (IQ 14–25 g/day) at baseline (Table 2) to a median of 68 g/day (IQ 66–75 g/day) during the HCD (P < 0.001) (Table 3), whereas it remained stable, with a median of 21 (IQ 18–22) g/day during the WSD (P = 0.38) (Table 3). The key foods provided a 7-fold difference in the RS concentration between the WSD (2.8 g/day) and HCD (20.7 g/day) and an almost 4.5-fold difference in the AX concentration between the WSD (3.6 g/day) and HCD (16.0 g/day) (Table 3). 10.1371/journal.pone.0159223.t003Table 3BODY.COMPOSITION OF TOTAL DIETARY INTAKE DURING THE WESTERN-STYLE DIET (WSD) AND THE HEALTHY-CARBOHYDRATE DIET (HCD).: WSD (n = 17)aHCD (n = 17)aWSD vs. HCDbMedianIQ rangeTotal rangeMedianIQ rangeTotal rangeP-valueEnergy (KJ) 9217 8450–10611 6855–14516 8412 7899–11228 6566–16799 0.97 Total CHOs (g) 300 271–346 260–479 325 292–375 269–603 0.17 Digestible CHOs (g) c 281 253–322 242–434 244 224–334 204–487 0.07 Dietary fibre (g) 21 18–22 17–45 68 66–75 65–116 0.0007 Protein (g) 96 85–111 53–153 83 74–105 65–222 0.21 Fat (g) 62 54–82 36–105 63 42–76 37–148 0.50 IQ, interquartile; CHOs, carbohydrates. aCalculated as the sum of macronutrition according to food records and the daily ration of key foods, as determined by chemical analysis. bDifferences between the interventions compared by paired t-test, except for dietary fibre and protein, which were compared by Wilcoxon signed-rank test. cCalculated as: (total carbohydrates–dietary fibre according to food records) + (digestible carbohydrates determined by chemical analysis). The energy content was 12% higher in the WSD key foods than in the HCD key foods (Table 1) because of the unexpectedly high protein content of the WSD bread and pasta compared with the HCD equivalents (S1 Table). The differences in the energy and nutritional constituents of the key foods were diminished when the total dietary intake was taken into account (Table 3). The median energy need of the subjects was determined to be 10,605 kJ (total range 7,992–13,402 kJ) per day, and the energy from the key foods in the HCD and WSD provided the subjects with 44% and 50% of their calculated median energy needs, respectively. The subjects’ body weights, waist circumferences and body fat percentages remained unchanged throughout the study (S2 Table). BODY.FAECAL CHARACTERISTICS AND GASTROINTESTINAL SYMPTOMS: Bowel movements over the three-day registration period rose from a median of 4 (total range 1–8) during the WSD to 5 (total range 2–13) (P < 0.01) during the HCD. The faecal consistency differed significantly (P = 0.02) between the two interventions. Six subjects reported constipation during the WSD compared with one subject during the HCD. Constipation difficulties were addressed by guidance on increased water intake and exercise, and no laxatives were necessary. Flatulence (P < 0.001) and stomach rumbling (P = 0.05) were increased during the HCD, but no other abdominal symptoms or adverse effects were reported. The diets were well tolerated and did not influence the subjects’ health concerns or abdominal symptom severity. Self-reported wellbeing was rated as moderately higher during the HCD compared with the WSD (P = 0.05). BODY.FAECAL CARBOHYDRATE RESIDUES: There were no differences in the faecal concentrations of carbohydrate residues (dry matter basis (DM)) during the pre-WSD and pre-HCD periods (Table 4). Consumption of the WSD was followed by a decline in total faecal DF from 17.2% DM to 12.3% DM (P = 0.01), whereas the opposite was the case for the HCD, which resulted in an increase from 18.4 to 24.2% DM (P = 0.002) (Table 4). Thus, faecal DF was 2 fold (95% CI 2-3-fold) (P < 0.0001) higher after HCD consumption compared with WSD consumption. In addition, the HCD increased RS residues from 2.3% DM to 3.0% DM compared with 0.8% DM after the WSD (P = 0.003), while AX residues were increased to 8.0% DM following the HCD compared with 3.3% DM after the WSD (P < 0.0001). 10.1371/journal.pone.0159223.t004Table 4BODY.FAECAL CARBOHYDRATE RESIDUES (% OF DRY MATTER) DURING THE PRE-PERIODS AND AFTER CONSUMPTION OF THE HEALTHY-CARBOHYDRATE DIET (HCD) COMPARED WITH THE WESTERN-STYLE DIET (WSD).: Pre-WSD (n = 19)WSD (n = 19)Pre-HCD (n = 19)HCD (n = 19)HCD vs. WSDMean95% CIMean95% CIMean95% CIMean95% CISEMP-valueRSDMSOa 2.1 1.2–2.9 0.8 0.3–1.2 2.3 0.7–3.8 3.0 1.4–4.7 - 0.003 NSPs 15.1 11.6 16.1 21.2 1.29 < 0.0001 Cellulose 4.2 3.2 4.1 4.7 0.37 0.002 Arabinoxylan 4.6 3.3 4.7 8.0 0.55 0.0001 Arabinose 2.1 1.5 2.2 3.8 0.26 < 0.0001 Xylose 2.5 1.8 2.5 4.1 0.30 < 0.0001 A:X ratio 0.84 0.86 0.88 0.93 0.04 0.29 Rhamnosea 0.5 0.5–0.6 0.6 0.5–0.7 0.5 0.5–0.6 0.6 0.5–0.7 - 0.07 Fucosea 0.2 0.2–0.2 0.2 0.2–0.3 0.2 0.2–0.2 0.2 0.2–0.2 - 0.25 Mannose 0.6 0.6 0.6 0.5 0.05 0.32 Galactose 1.3 1.3 1.3 1.8 0.08 < 0.0001 Glucosea 3.1 2.2–3.9 1.9 1.4–2.3 3.9 2.0–5.8 4.5 3.1–5.8 - 0.0001 Uronic acid 0.8 0.7 0.8 1.0 0.07 0.002 Dietary fibrea 17.2 14.5–19.8 12.3 10.2–14.5 18.4 14.3–22.6 24.2 20.6–27.9 - < 0.0001 A:X ratio, arabinose to xylose ratio; CI, confidence intervals; NSPs, non-starch polysaccharides; RSDMSO, resistant starch; SEM, standard error of the mean (ANOVA for repeated measurements using subject, diet, period and baseline values as covariates). aData were logarithmically transformed before data analysis and therefore, 95% CI are given. BODY.FAECAL MICROBIAL COMPOSITION: 16S rRNA gene amplicon sequencing was performed to identify the microbial compositions in faecal samples collected at baseline and at the end of the interventions. An average of 59,113 sequencing reads was obtained for each sample after quality filtering, including an average of 863 OTUs per sample. The number of observed species (alpha-diversity) was significantly lower in the stools collected after the HCD (an average of 615 species) compared with those collected after the WSD (675 species) (P < 0.0001). This difference in species richness was in agreement with the Chao1 (P < 0.05) and phylogenetic diversity (P < 0.005) alpha-diversity metrics. The HCD also resulted in a significant change in the beta-diversity of the faecal microbiota, as shown by principal coordinates analysis of the weighted UniFrac distance metric (Fig 2A). The effects of the HCD were observed both within and between subjects (Fig 2B). Specifically, the microbiota were more heterogeneous, as indicated by the larger weighted UniFrac distances after HCD consumption compared with the baseline values for individual subjects (Fig 2B, “Within Subjects”), as well as for all subjects combined (Fig 2B, “Between Subjects”). 10.1371/journal.pone.0159223.g002Fig 2BODY.A PRINCIPAL COORDINATE ANALYSIS OF WEIGHTED UNIFRAC VALUES BETWEEN FAECAL MICROBIAL COMMUNITIES.: The green and red arrows represent bacterial communities before and at the end of healthy-carbohydrate diet (HCD) and Western-style diet (WSD) consumption, respectively. The dotted lines connect the microbiota present during consumption of the two baseline diets for each individual. B Box plot of the weighted UniFrac distances. Intra-individual microbiota distances are shown on the left, and inter-individual distances are shown on the right. * P< 0.05, **** P< 0.0001 (Wilcoxon signed-rank test). The proportion of Bifidobacterium in the faecal microbiota was significantly increased after HCD consumption compared with that detected after WSD consumption (Fig 3). Non-significant increases were also observed in Lactobacillus, Clostridium, and Akkermansia. Conversely, the HCD resulted in reductions in the proportions of the Bacteroidetes genera Bacteroides, Parabacteroides, Butyricimonas, Odoribacter, and Paraprevotella (Fig 3). The proportions of five genera in the Firmicutes phylum were also lower following consumption of the HCD, including certain Ruminococcus species (Fig 3). These Ruminococcus species are members of the Lachnospiraceae family and are not related to Ruminococcus bromii, another bacterial species associated with consumption of DF.[14, 17–19] The proportions of certain members of Proteobacteria were also decreased following the HCD compared with the WSD, including Sutterella and members of the Desulfovibrionaceae family. 10.1371/journal.pone.0159223.g003Fig 3BODY.BOX PLOT OF THE LOG:2-transformed fold-changes in the relative abundances of taxa in the faeces collected at the end of consumption of the healthy-carbohydrate diet (HCD) compared with that of the Western-style diet (WSD). Only taxa that were significantly affected by diet (P < 0.05) are shown. Family, order, and genus distinctions are represented by (f), (o), and (g), respectively. The boxes and text are coloured according to the phyla as follows: Actinobacteria (blue), Bacteroidetes (green), Firmicutes (purple), and Proteobacteria (red). * P < 0.05, ** P < 0.01, *** P < 0.001 (Wilcoxon signed-rank test). BODY.FAECAL SCFAS AND LACTIC ACID: The SCFA concentrations during the two run-in periods were similar. The diets caused significant differences in SCFA concentrations, as illustrated in Fig 4A. During the WSD (from weeks 0 to 4) both acetate and butyrate declined 19% (95% CI 5–30%) (P = 0.01) and 37% (95% CI 22–50%) (P < 0.001), respectively. Conversely, acetate (P = 0.12) and butyrate (P = 0.68) remained unchanged during the HCD. A large range was observed in the pre-HCD concentrations, from 1.3 to 27.6 mmol/kg. The butyrate concentration increased in response to the HCD in half of the subjects, mainly those with the lowest pre-HCD levels, whereas the subjects with high pre-HCD concentrations experienced a decline in the concentration after HCD consumption (Fig 4B). The concentration of lactic acid was not affected by the diet (P = 0.18). 10.1371/journal.pone.0159223.g004Fig 4BODY.FAECAL SHORT-CHAIN FATTY ACID (SCFA) AND BRANCHED-CHAIN FATTY ACID (BCFA) CONCENTRATIONS.: A, C Differences between Western-style diet (WSD) and healthy-carbohydrate diet (HCD) concentrations are indicated by NS; non significant, *P < 0.05 and **P< 0.01 (ANOVA for repeated measurements using subject, diet, period and baseline values as covariates). The values are the medians with interquartile ranges (n = 19) as the data were log-transformed. B Individual faecal butyrate concentration before (Pre-HCD) and after HCD. The solid line represents the median pre-HCD concentration and the dashed lines indicate the interquartile range of the pre-HCD concentrations. The total concentration of branched-chain fatty acids declined 30% (95% CI 3–50%) (P = 0.03), that of isobutyrate decreased 28% (95% CI 1–48%) (P = 0.05) and that of isovalerate decreased 21% (95% CI 6–52%) (P = 0.03) after the HCD compared with the WSD (Fig 4C). The caproic acid concentration rose slightly (from a median of 0.5 to 1.1 mmol/kg (P = 0.001)) during the HCD. The concentrations of formic acid, valeric acid, heptanoic acid and succinic acid concentrations remained unchanged, and those of sorbic acid, benzoic acid and hippuric acid were all below the detection levels. BODY.DISCUSSION: In this 4-week, randomised, crossover, dietary intervention study of individuals with MetS, we found that a diet rich in RS and AX modified the gut microbiome and increased the concentration of faecal SCFAs in general and those of butyrate and acetate in particular compared with a low-fibre diet. The HCD conferred a significant decrease in bacterial species richness in all individuals. This finding corroborates that of a previous study reporting that a diet high in RS is associated with decreased bacterial diversity relative to other diets.[52] However, we also found that HCD consumption increased the inter- and intra-individual variation of the intestinal microbiota. This finding indicates that the enrichment of specific populations of bacteria in the intestine by DF is both diet- and subject-dependent.[18, 19] This effect of DF was significant because unlike other human dietary intervention studies,[52, 53] the intra-individual differences in the intestinal microbiota were typically as large as those found between subjects. This outcome is probably due to the strong selection of certain bacteria by DF of the taxa that were already present in the intestines of each individual prior to the HCD intervention.[19] In addition to dietary factors, the intestinal microbiota is influenced by e.g. decreased diversity in elderly subjects, immunological factors, antibiotic treatment, genetic heritage and microbial exposition at birth.[9] The range from 39 to 75 years of age in the subjects may contribute to the between- subject differences. The predominant change in the faecal microbiota with the introduction of the HCD was the enrichment of members of Bifidobacterium, a genus regarded to be beneficial to human health.[54] The capacity of DF to promote Bifidobacterium has been found in many studies.[14, 15, 55] Bifidobacterium is a saccharolytic bacterium that produces acetate and lactic acid as a result of fermentative growth.[56, 57] Hence, the increased faecal acetate concentration observed with HCD consumption may have been the result of stimulation of Bifidobacterium growth and metabolism by DF in the gut. Acetate production by Bifidobacterium confers protection against gastrointestinal pathogens[58, 59] and it may be consumed by butyrate-producing bacteria in the gut.[60, 61] An in vitro study has also found that up to 90% of produced butyrate is derived from acetate.[62] The explanation why we did not find a diet effect on lactic acid may be that it is readily fermented whereby it also acts as a precursor for butyrate synthesis.[63] Therefore, the increased faecal butyrate concentration is most likely the result of increased acetate and lactic acid produced by Bifidobacterium rather than stimulation of butyrate-producing microorganisms. Conversely, Firmicutes, Bacteroidetes and Proteobacteria were significantly reduced by the HCD compared with the WSD. WSD consumption, on the other hand, enhanced the proportions of Bacteroides, Sutterella and Ruminococcus. The specific Ruminococcus species enriched by the WSD are members of the Lachnospiraceae family, which includes species previously associated with inflammatory bowel disease.[64] In the present study, we were unable to differentiate between the contribution of AX and that of RS to the enhanced SCFA production. AX derived from whole-grain rye has been demonstrated to cause a significant increase in the butyrate concentration in humans,[3] and AX derived from whole-grain rye and enzyme-treated wheat bran has been found to be superior to RS type 2 with regard to butyrate and acetate production in pigs.[15, 30] However, a concentrate of AX from wheat has been demonstrated not to affect the colonic butyrate concentration or the caecal digesta butyrate pool in pigs.[65] The reasons for these differences in butyrate response among these studies cannot be determined with certainty but may potentially be related to the manner by which AX was provided, for example, as a concentrate from wheat[66] or as part of a whole-grain matrix.[3, 16] Fermentation of an AX concentrate can be expected to be rapid and accompanied by a pH drop, mainly in the caecum,[66] whereas the fermentation of AX from a whole-grain matrix will be slower, with pH drops not only in the caecum but also in the proximal and mid colon.[20] In the present study, this situation was even more complex because the RS provided from raw potato starch was degraded more rapidly than that from high-amylose corn starch, which was degraded at a slower rate at more distal locations.[20] Based on the results of Nielsen et al.[15], we speculate that the RS from high-amylose corn starch was responsible for the higher RS levels found in the faeces after HCD consumption. Surprisingly, the diet-induced differences in the SCFA concentrations observed in this study were mainly due to declining SCFA concentrations during the WSD, despite the significantly increased consumption of total DF in the HCD compared with that in the WSD and at baseline. Furthermore, the faecal DF, RS and AX residues increased during the HCD and declined during the WSD, indicating good dietary adherence during the study. One possible explanation for the changes in the SCFA concentrations observed during the interventions might be that the subjects had a higher habitual intake of DF than that registered in the food records because rye bread in particular is a normal part of the Danish diet. In addition, food data obtained from food records may be underreported.[66] Still, Jonsdottir et al.[67] found a mean DF intake of 22 g/d in a Nordic population with MetS, which is similar to the reported baseline intake among the subjects reported here. A second explanation may be that the subjects were instructed to limit their intake of fibre-rich fruits, nuts and vegetables during the interventions, and it is likely that such limited intake contributed to their changes in intestinal microbial environment and SCFA production. A third explanation may be that we underestimated the effect of the HCD on total colonic SCFA production because we did not consider potentially increased faecal output from DF. Recently, it has been shown that diets rich in RS or in AX increase both the production and faecal output of total SCFAs, particularly acetate and butyrate, in pigs.[15] We did find a significant increase in bowel movements during the HCD, pointing to increased faecal output. A noteworthy finding was the decreased concentration of BCFAs in the faeces during the HCD. This finding indicates reduced protein fermentation, which counteracts the accumulation of potentially harmful metabolites.[68] Bacteroides, an abundant human gut resident[69] capable of proteolytic fermentation[56] and BCFA production,[70] was down-regulated by the HCD, which corroborates these results. Cummings et al.[71] have also reported decreased BCFAs after RS-enriched diet consumption. Consumption of a diet rich in AX has also been shown to lower caecal digesta p-cresol, indicating reduced protein fermentation.[65] A limitation of the present study is that the dietary compositions were based on nutrition labelling, but chemical food analyses performed post hoc disclosed that the key foods had a higher protein content in the WSD than in the HCD, which could have resulted in differences in the BCFAs. Nevertheless, these differences were evened out when total dietary intake was taken into account, causing the difference in total protein intake between the groups to lose significance, and only DF intake was significantly different in favour of the HCD. In conclusion, the results of our study support the hypothesis that high intake of both RS and AX is capable of changing the intestinal microbiota and SCFA production in subjects with MetS in contrast with a low-fibre WSD. Most distinctly, Bifidobacterium was clearly enriched by the HCD, which was in strong agreement with the increased faecal acetate concentration. Also, dysbiotic changes observed during the WSD emphasise the need for balanced diets, including DF from various sources. However, long-term randomised controlled intervention studies are needed to investigate the effects of DF on the microbiota and SCFA production in a more continuous setting. In addition, further attempts to compare the impacts of different DFs on metabolic risk factors and intestinal mucosal function are needed. BODY.SUPPORTING INFORMATION: S1 InformationBODY.STUDY PROTOCOL.: English translation and the original version in Danish. (DOCX) Click here for additional data file. S2 InformationBODY.CONSORT CHECKLIST.: (DOC) Click here for additional data file. S1 TableBODY.NUTRITIONAL COMPOSITION OF THE KEY FOODS ON A DRY MATTER (DM) BASIS FOR THE HEALTHY-CARBOHYDRATE DIET (HCD) AND WESTERN-STYLE DIET (WSD).: (DOCX) Click here for additional data file. S2 TableBODY.BODYWEIGHT, BODY FAT PERCENTAGE AND WAIST CIRCUMFERENCE AT RUN-IN AND AT THE END OF THE HEALTHY-CARBOHYDRATE DIET (HCD) AND WESTERN-STYLE DIET (WSD).: Values are presented as medians and interquartile ranges in brackets. (DOCX) Click here for additional data file.

TITLE: Inhibitory effects of intravenous lansoprazole 30 mg and pantoprazole 40 mg twice daily on intragastric acidity in healthy Chinese volunteers: A randomized, open-labeled, two-way crossover study ABSTRACT.SUMMARY: ABSTRACT.BACKGROUND: Until now there has been no study that directly compares the effect of lansoprazole and pantoprazole administered intravenously on intragastric acidity. The aim of this study is to compare the effect of lansoprazole (30 mg) and pantoprazole (40 mg) administered intravenously on gastric acidity. ABSTRACT.MATERIAL/METHODS: Helicobacter pylori-negative healthy volunteers were recruited in this open-label, randomized, two-way crossover, single centre study. Lansoprazole at 30 mg or pantoprazole at 40 mg was intravenously administered twice daily for 5 consecutive days with at least a 14-day washout interval. Twenty-four-hour intragastric pH was continuously monitored on days 1 and 5 of each dosing period. ABSTRACT.RESULTS: Twenty-five volunteers completed the 2 dosing periods. The mean intragastric pH values were higher in subjects treated with lansoprazole than those with pantoprazole on both day 1 (6.41±0.14 vs. 5.49±0.13, P=0.0003) and day 5 (7.09±0.07 vs. 6.64±0.07, P=0.0002). Significantly higher percentages of time with intragastric pH >4 and pH >6 were found in the subjects treated with lansoprazole than those with pantoprazole on day 1 (pH >4, 87.12±4.55% vs. 62.28±4.15%, P=0.0012; pH >6, 62.12±4.12% vs. 47.25±3.76%, P=0.0216) and pH >6 on day 5 (76.79±3.77% vs. 58.20±3.77%, P=0.0025). ABSTRACT.CONCLUSIONS: Intravenous lansoprazole produces a longer and more potent inhibitory effect on intragastric acidity than does intravenous pantoprazole. BODY.BACKGROUND: Proton pump inhibitors (PPIs) have been used widely and achieved great benefits in managing acid-related upper gastrointestinal diseases, including gastroesophageal reflux disease (GERD), peptic ulcer disease, Helicobacter pylori infection, nonsteroidal anti-inflammatory drug (NSAID)-associated gastric mucosal damage, hypersecretory states such as Zollinger-Ellison syndrome, non-variceal upper gastrointestinal bleeding, and even functional dyspepsia. The pathophysiologic basis of these management benefits lies in their potent gastric acid inhibitory effects. Intravenous administration of a PPI, which usually provides gastric acid suppression faster than oral administration [1], is mostly recommended for patients with non-variceal upper gastrointestinal bleeding, with acid-related diseases, who cannot tolerate oral intake, or those with a disorder of swallowing [2]. There have been many studies that compared the effects of different PPIs through oral administration, but only 1 study evaluated the effect of different PPIs on the intragastric acidity in healthy adults through intravenous administration (esomeprazole [40 mg] vs. lansoprazole [30 mg]) [3]. However, there has been no study that directly compares the efficacy of intravenous lansoprazole and pantoprazole in terms of inhibiting intragastric acidity. The aim of this study was to evaluate the inhibitory effect of intravenous lansoprazole (30 mg) and pantoprazole (40 mg) twice-daily for 5 consecutive days on intragastric acidity in healthy Chinese volunteers. BODY.MATERIAL AND METHODS: BODY.SUBJECTS: BODY.INCLUSION AND EXCLUSION CRITERIA: Healthy male or non-pregnant female volunteers aged 18–45 years, with a body mass index (BMI) of 19–25 kg/m2 and with extensive metabolizer (EM) status for CYP2C19 genotypes, were included. Subjects who had a history of a severe disease in any major organ (eg, renal, hepatic or cardiovascular disease) that might affect the pharmacokinetics of PPIs were excluded. Subjects who had current or past (within 6 months prior to the screening) endoscopic evidence of esophageal pathology or a history of gastric or esophageal surgery, who took PPIs, and NSAIDs or any other drugs that may cause injuries to the gastric mucosa within 2 weeks before the first dose of the study drug, and who would require any concomitant drugs during the study, were excluded. Subjects who had a history of significant clinical illness, drug or alcohol abuse, and any conditions that could modify the absorption of the study drugs as judged by the investigators within the 2 weeks before the first dose of study drugs were also excluded. The study was performed according to the ethical principles of the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Changhai Hospital, Shanghai, China. All subjects gave written informed consent prior to being enrolled in the trial. BODY.STUDY DESIGN: The study was an open label, randomized, 2-way crossover design, and performed at 1 center. An initial screening visit took place within 14 days prior to the first study day and consisted of a complete medical history, physical examination and measurement of laboratory safety variables such as renal and hepatic functions, as well as the urine pregnancy test for female subjects. In addition, CYP2C19 genotypes and the status of H. pylori infection were determined as described below. Eligible subjects were randomized to receive either lansoprazole (Jiangsu Aosaikang Pharmaceutical Co. Ltd, Nanjing, China) at 30 mg or pantoprazole (Nycomed GmbH, Konstanz, Germany) at 40 mg via intravenous infusion within 30 min twice daily at 8:00 am and 8:00 pm on day 1 through day 5. Then, after a washout interval of 14–21 days, the subjects were switched to receive another PPI (pantoprazole or lansoprazole, where appropriate), in the same fashion as described above. The subjects took visits 2–12 days before the first dosing period and 5–7 days after each dosing period. Standardized meals, not high in fat or calories, were provided by the research center from day 1 through day 5. Meals were administered in an identical fashion during both dosing periods. Alcohol and caffeinated beverages, and any new or intensified physical activities were not permitted during the study period until the completion of the last follow-up visit. On days 1 and 5 of each of the dosing periods, 24-h intragastric pH was monitored as described below. BODY.MEASUREMENT OF INTRAGASTRIC PH: After a 12-h fast, 24-h intragastric pH was recorded starting from 8 am after the first dose on day 1 and day 5 of each dosing period using a pH-sensitive microelectrode (Medtronic, Copenhagen, Denmark) linked to a Digitrapper MKIII recording system (Medtronic). The electrode was inserted trans-nasally and placed about 8–10 cm below the lower esophageal sphincter as identified by a sharp decrease in pH indicating the point at which the electrode crossed the sphincter. There were marks on the surface of the catheter to identify the position. Using a microprocessor, this unit was able to record the subject’s intragastric pH over the 24-h period. Then, the Digitrapper™ data were downloaded onto a personal computer to calculate the percentage of time in which the intragastric pH was >4 and percentage of time in which the intragastric pH >6, along with the 24-h median intragastric pH. A 2-point calibration of the probe was made before each recording, using standard buffers of a pH of 7.01 and pH of 1.07. All the subjects stayed in the research center from 8:00 pm on the day prior to pH monitoring. At 6:00–7:00 am of the pH monitoring day, the pH electrode was placed 30 min before the first dosage and the measurement of the intragastric pH values started at the same time of the first dosage, and continued until 8 am on the next day. The pH monitoring interval continued at least 22 hours. The subjects were mobile and were instructed not to lie down for longer than 10 min each time during the day and lie down to sleep at 10 pm at night until 6 am next morning. Subjects were prohibited from eating or drinking after 8 pm of the monitoring day. BODY.ASSESSMENT OF CYP2C19 GENOTYPES AND :H. pylori infection Metabolizer status for CYP2C19 genotypes was assessed by polymerase chain reaction (PCR) technique as previously described [4]. Briefly, 0.2 ml blood was used for DNA extraction by QIAamp DNA Blood Mini Kits (QIAGEN Company); 100 ng DNA was amplified in the first round of PCR in a 25 μl reaction volume containing 1.5 mmol/L MgCl2, 1×PCR buffer (Takara, Dalian, China), 0.5 U Hot-start Taq (Takara), 200 μmol/L dNTP, 1 μmol/L primers: 5′ CCATTATTTAACCAGCTAGG 3′ and 5′CCATTATTTAACCAGCTAGG 3′ at the following condition: denaturation at 95°C for 5 min and followed by 30 cycles of 94°C for 30 s, 60°C for 1 min, 72°C for 2 min and then extension at 72°C for 7 min. 1 μl product of the first round of PCR was then subjected to the second round of PCR with 1 μmol/L primers for the exon 4 of CYP2C19 (5′ TCTGTTAACAAATATGAAGTGTT3′ and 5′ TCTAGGCAAGACTGTAGTATTC3′) and primers for the exon 5 of CYP2C19 (5′ TTGGCATATTGTATCTATACCTT3′ and 5′ CTAGTCAATGAATCACAAATAC3′). The second round of PCR was performed under the following conditions: denaturation at 95°C for 5 min and followed by 30 cycles of 94°C for 30 s, 53°C for 1 min, 72°C for 2 min and then extension at 72°C for 7 min. The products were then separated by 1% gel electrophoresis and subjected to DNA sequencing analysis (Invitrogen Corporation). H. pylori infection status was detected by a 13C-labeled urea breath test within 7 days before the enrollment. This test has been reported to have high sensitivity, specificity and accuracy [5,6]. BODY.SAFETY ANALYSIS: Physical examinations, abdominal ultrasonography and electronic cardiograms were conducted and blood samples collected 2 days before and 5–7 days after each dosing period. All reported or observed adverse events such as symptoms, signs, abnormal laboratory testing, abdominal ultrasonography or electronic cardiograms were recorded. BODY.STATISTICAL ANALYSIS: All data were analyzed using SAS 9.1 (SAS Institute Inc., Cary, NC, USA). ‘Per protocol’ population was used in the efficacy analysis, which included all subjects who satisfied the inclusion/exclusion criteria and complied with study drug administration per protocol, and had no major protocol deviations that might affect intragastric pH. The primary efficacy outcome was analyzed by comparing the percentage of time that the intragastric pH was higher than a threshold of 4 and 6 between treatment groups on days 1 and 5. The percentage of time in which the pH >4 and pH >6 over the 24-h period and median 24-h intragastric pH were analyzed using a mixed model ANOVA with fixed effects for period, sequence and treatment and a random effect for subjects within sequence. The mean value for each treatment and mean treatment differences (lansoprazole vs. pantoprazole) were estimated with least-squares mean (LSM) percentages ± standard error of means (SEM). The data on day 1 and 5 were analyzed. The percentage of subjects with intragastric pH above 4 for more than 12 h and pH above 6 for more than 16 h on day 1 and day 5 was analyzed using Fisher’s exact test. The safety data from all subjects who received at least 1 dose of study medication were summarized descriptively, and analyzed by the chi-square test. A P value of less than 0.05 was considered statistically significant. BODY.RESULTS: BODY.SUBJECTS: Twenty-three (13 males and 10 females, ages ranging between 21 and 43 years) H. pylori-negative healthy volunteers were enrolled in the study. All completed the 2 dosing periods. The subjects were divided into 2 groups; 12 and 11 received intravenous lansoprazole and pantoprazole at the first dosing period, and then intravenous pantoprazole and lansoprazole during the second dosing period, respectively. There were no differences in the sex distribution (8/4 vs. 5/6 males/females), age (28.92±6.97 vs. 24.45±2.54 years), or BMI (21.95±1.39 vs. 21.73±1.51 kg/m2) between the 2 groups. BODY.INTRAGASTRIC PH: The mean intragastric pH values in each hour on day 1 and day 5 are presented in Figures 1 and 2. Generally, on both day 1 and day 5, after the first dosing at 8 am, the pH value increased, with a transient drop starting at 1.5 h, and then increased again at 3.5 h. At 6.5 h after the dosing, the pH value dropped and continued to drop until at 9.5 h, when the pH value rebounded. After the second dosing at 8 pm (12 h after the first dosing), the pH values dropped slightly, but maintained at levels higher than those at the beginning of the dosing. There were significant differences in the pH values at the time points, between hours 2–14 on day 1, and the time points in hours 1–5, 21 and 22 on day 5. The percentages of time with intragastric pH >4 and pH >6 over the first 4 h and 24 h on day 1 and 5 are presented in Table 1. Significantly higher percentages of time with intragastric pH >4 and pH >6 were observed in the subjects treated with lansoprazole than in those treated with pantoprazole during the first 4 hours, on day 1 and pH >6 on day 5. The mean time for pH >4 and pH >6 with lansoprazole was shorter than with pantoprazole on day 1, but the difference was not statistically significant (109.61±32.15 min vs. 153.42±29.36 min, P=0.3304 for pH 4; and 211.93±39.18 min vs. 288.43±35.79 min, P=0.1706 for pH 6). The percentages of subjects with intragastric pH>4 for at least 12 hours were 94% and 83% for lansoprazole and pantoprazole, respectively, on day 1, and 100% for both lansoprazole and pantoprazole on day 5. The percentages of subjects with intragastric pH>6 for at least 16 hours were 31% and 13% on day 1, and 67% and 43% on day 5 for lansoprazole and pantoprazole, respectively. BODY.SAFETY AND TOLERABILITY: Both lansoprazole and pantoprazole, intravenously administrated, were well tolerated. A drug-related moderate skin rash occurred in a female volunteer after a dose of pantoprazole, and mild somnolence which might be related to lansoprazole occurred after a dose of lansoprazole. These adverse events did not result in drug discontinuation, and disappeared without any medication. No serious adverse events occurred. There were no clinically significant abnormal laboratory, ultrasonographic and electrocardiographic findings. BODY.DISCUSSION: Maintenance of the intragastric pH above 4 is crucial for preventing stress-related upper gastrointestinal bleeding [7–9]. Moreover, the recent guidelines and consensus have recommended elevating the intragastric pH above 6 with intravenous high-dose PPIs in the treatment of non-variceal gastrointestinal bleeding [2,10–14]. This recommendation is based on the evidence that the acidic environment does not benefit to the coagulation, and neutralizing the intragastric acidity is required for successful coagulation. Laterre et al. [15] reported that a large intravenous bolus dose (80 mg) of omeprazole, followed by 8 mg per h, achieved more than 80% of the dosing period with pH above 6. In addition, van Rensburg et al. [16] reported that in patients with acute bleeding peptic ulcers, an intravenous bolus injection of 80 mg of pantoprazole after successful endoscopic hemostasis, followed by continuous infusion of either 6 mg/h or 8 mg/h pantoprazole for 72 h, rapidly increased the intragastric pH to values of above 6. A number of trials have compared the effects of different PPIs on gastric acidity in healthy volunteers, patients on aspirin or NSAIDs and those with pathological conditions. It has been shown that the relative potencies of the 5 available PPIs compared to omeprazole were 0.23, 0.90, 1.00, 1.60, and 1.82 for pantoprazole, lansoprazole, omeprazole, esomeprazole, and rabeprazole, respectively, in the population with GERD [17]. Compared with healthy volunteers, patients with GERD needed a 1.9-fold higher dose and H. pylori-positive individuals needed only about 20% of the dose to achieve a given increase in mean 24-h intragastric pH. However, these data are based on clinical trials using oral administration, which may not reflect the situation where the PPIs are administrated intravenously. There have been clinical trials evaluating the effect of PPIs on intragastric acidity; however, there have been no studies comparing, head-to-head, the effect of intravenous pantoprazole and lansoprazole in the same clinical study. In the present study, we conducted an open-label, randomized, 2-way crossover, single centre clinical trial, which was the first to date to compare the effect of lansoprazole (30 mg) and pantoprazole (40 mg) administered intravenously twice daily for 5 consecutive days on gastric acidity. Pantoprazole is one of the most widely used PPIs in the world. Several previous studies evaluating whether oral or intravenous pantoprazole is a consistent and long-lasting inhibitor of gastric acid secretion have achieved satisfactory results in patients with acid-related diseases [18,19]. Numerous studies and meta-analyses have confirmed the efficacy of intravenous pantoprazole in treating peptic ulcer bleeding and stress ulcer bleeding, and thus pantoprazole is now one of the PPIs most used intravenously in treating non-variceal gastrointestinal bleeding. Lansoprazole is also a newly developed PPI with high effectiveness in controlling intragastric acid and in treating acid-related upper gastrointestinal diseases [20–26]. Several clinical trials have been conducted to compare the effect on intragastric acid control by oral lansoprazole with that by either oral or intravenous pantoprazole in healthy volunteers and patients with GERD [27–30]. Almost all the results showed that oral lansoprazole is stronger or equal to oral or intravenous pantoprazole in controlling intragastric acid. Huang et al. [30] reported a crossover trial of 74 healthy male volunteers who received oral lansoprazole 30 mg or pantoprazole 40 mg once daily for 5 consecutive days with at least a 2-week washout period between the regimens. It was found that compared with pantoprazole, lansoprazole produced higher mean 24-h intragastric pH values, greater proportions of time in which the intragastric pH was above 3, 4 and 5 on day 1, a higher mean 24-h intragastric pH value, and a greater percentage of time that the intragastric pH was above 4 on day 5. Freston et al. [28] reported an open label, 2-way crossover, and single-center study comparing the effect of lansoprazole at 30 mg per nasogastric tube with that of pantoprazole at 40 mg intravenously for 5 consecutive days. Similarly, it was found that lansoprazole produced higher mean 24-h intragastric pH values relative to pantoprazole on day 1 and day 5. Lansoprazole sustained the intragastric pH above 3 (days 1 and 5), 4, and 5 (day 1) longer than pantoprazole. Although previous trials [31,32] have demonstrated that the effects on intragastric acidity with lansoprazole by oral and intravenous routes are similar, and compared with pantoprazole in healthy volunteers or patients with pathological conditions, oral lansoprazole produced a more potent ability in inhibiting gastric acid secretion. Intravenous formulation is still the first choice for acute non-variceal gastrointestinal bleeding. In addition, some patients requiring acid suppression may be unable to take oral medications due to dysphagia. Therefore, in many cases intravenous lansoprazole cannot be replaced by its oral formulation. However, there has been no study that directly compares the effect on intragastric acidity between intravenous lansoprazole and intravenous pantoprazole. The present study was the first to explore this issue. In the present study, our findings are in agreement with the results of previous trials and found that lansoprazole produced significantly higher mean intragastric pH values and percentages of time with intragastric pH >4 and pH >6 than pantoprazole on day 1 and pH >6 day 5. The importance of the CYP2C19 genotype in acid suppression by PPIs has been elucidated [33]. EMs and poor metabolizers (PMs) of this isoenzyme differ in their elimination of drugs (such as PPIs) that depend on it for clearance. Therefore, the genetic difference in drug metabolism can cause a wide interindividual variation in antacid effect of PPIs (ie, PMs are associated with a stronger effect than Ems). For example, the researchers noted that therapeutic efficacy of PPIs is strongly linked to interpatient differences in drug metabolism [33]. In the present study, we only analyzed the inhibitory effect of PPIs on gastric acid secretion in volunteers with EMs. Thus, the results of the present study may be relevant in the populations with a low prevalence of PMs, such as in the Chinese, where the reported prevalence of PMs is about 12.7% [34], but may not be applicable to other populations where the prevalence of PMs is high, which is a major limitation of this study. In addition, in clinical practice the patients likely to receive intravenous PPI treatment are those in a critical care setting and fasting conditions. The present study was conducted with healthy volunteers who did not accurately reflect the demographic characteristics and comorbidities present in critically ill patients in intensive care units. The efficacy of intravenous lansoprazole in clinical settings needs further study. BODY.CONCLUSIONS: Both pantoprazole and lansoprazole administered intravenously produced strong inhibition of gastric acid control, while lansoprazole administered intravenously significantly improved the percentage of time with intragastric pH >4, the median 24-hour intragastric pH on day 1 and day 5 relative to pantoprazole. The results of the present study indicate that lansoprazole administered intravenously is more effective in maintaining high intragastric pH in healthy subjects than is pantoprazole, and offers the potential to treat acute non-variceal upper gastrointestinal bleeding.

TITLE: Long-term results of a randomized controlled trial evaluating preoperative chemotherapy in resectable non-small cell lung cancer ABSTRACT.OBJECTIVE: We aimed to evaluate whether preoperative chemotherapy provides benefits in the survival and prognosis of patients with non-small cell lung cancer (NSCLC) in resectable stages I to IIIA, except T1N0. ABSTRACT.METHODS: In this randomized, controlled trial, 356 patients with stage I (except for T1N0), II and IIIA NSCLC were assigned to either the preoperative chemotherapy plus surgery arm (179 patients) or the primary surgery arm (177 patients). Both treatments were followed by adjuvant chemotherapy. The end point of this study included overall survival (OS), progression-free survival (PFS), and survival rate associated with clinical remission. ABSTRACT.RESULTS: Statistical survival difference was found between the preoperative chemotherapy plus surgery arm and the surgery-alone arm. However, the median survival time (MST) in the preoperative chemotherapy arm was lower than that of surgery-alone arm (MST, 45.42 months vs 57.59 months) (P = 0.016). When comparing the effect of preoperative chemotherapy at each stage of NSCLC, a statistical survival difference was found in stage II NSCLC but not in stage I and IIIA (MST 40.86 months vs 80.81 months) (P = 0.044). However, no statistically significant difference in PFS was noticed between the two arms, except for stage I NSCLC (hazard radio [HR] = 0.87; 95% CI, 0.561–1.629; P = 0.027). The survival rate was higher for patients who had clinical remission after preoperative chemotherapy, but the differences did not reach statistical significance (MST 42.10 months vs 35.33 months) (P = 0.630). ABSTRACT.CONCLUSION: Preoperative chemotherapy did not show benefits in OS and PFS for stage I–IIIA NSCLC patients. BODY.INTRODUCTION: As one of the leading causes of mortality throughout the world, non-small cell lung cancer (NSCLC) accounts for approximately 80% of the 1.18 million deaths from lung cancer each year.1,2 For patients with IB–IIIA stages of NSCLC, surgery is well recognized as the most effective treatment. However, 30% to 70% of patients who had tumor completely resected still experience relapse.3,4 Surgery followed by adjuvant chemotherapy is thus generally recommended to most patients with NSCLC. A meta-analysis in 1995 showed there was a 5% increase in 5-year survival with surgery followed by adjuvant chemotherapy compared with surgery alone (hazard ratio [HR], 0.87; P = 0.08).5 The International Adjuvant Lung Cancer Trial (IALT), which included 1867 patients, has reported a significant overall survival (OS) benefit for adjuvant chemotherapy.6 Preoperative chemotherapy also provides many benefits to cancer patients and is offered to shrink tumors before surgical resection, to eliminate micrometastases, and to assess chemotherapy response to identify patients who will potentially benefit from adjuvant chemotherapy.7,8 However, given conflicting results in the efficacy of preoperative chemotherapy from past clinical trials, the benefits of preoperative chemotherapy in NSCLC remain controversial. The latest meta-analysis evaluating preoperative chemotherapy in NSCLC, which included 3615 patients from 14 randomized, controlled trials (RCTs), showed that patients with stage IIIA NSCLC benefited the most from preoperative chemotherapy (B = −0.18; t = −5.25; P = 0.006),9 although several independent trials did not show benefits of preoperative chemotherapy for NSCLC patients.10 In this study, we analyzed the long-term results of a multicenter RCT to compare preoperative chemotherapy plus surgery vs surgery alone for patients with stage I to IIIA (except for T1N0) NSCLC. BODY.PATIENTS AND METHODS: BODY.PATIENT SELECTION: A total of 356 patients were enrolled in this trial from the Shanghai Chest Hospital, the Shanghai Pulmonary Hospital, and the Shanghai Huadong Hospital between February 1995 and October 2001. Eligible patients were required to have stage I (except for T1N0), II, or IIIA histologically confirmed NSCLC, according to the tumor-node-metastasis (TNM) classification. They had to be 75 years old or younger, have a Karnofsky performance score of 80 or higher, and have had curative surgical resection. Previous chemotherapy or radiotherapy was not allowed. Patients with other organic diseases were ineligible. Preinclusion investigations consisted of history and physical evaluation, chest X-ray, computed tomographic (CT) scan of the chest and brain, upper abdomen CT scan or ultrasound, and isotope bone scan. The institutional review boards of the participating centers approved the protocols used in this study. All patients provided written, informed consent. BODY.TREATMENTS: Patients were randomly assigned into either a preoperative chemotherapy plus surgery group or a surgery-alone group. Patients from both groups received adjuvant chemotherapy after surgery. The chemotherapy consisted of mitomycin (6 mg/m2), cisplatin (80 mg/m2), and vindesine (2.5 mg/m2) on day 1, 8, and 15. For both study arms, chemotherapy was repeated every 28 days for 4 cycles. In the preoperative chemotherapy arm, 1–2 cycles of chemotherapy treatment had to be completed before surgery. For both arms, 4–6 cycles of chemotherapy were given to patients after surgery. A physical examination and blood analyses were performed at each cycle during chemotherapy. Responses to chemotherapy were assessed according to the WHO criteria. Surgery had to start after randomization in the surgery-alone arm and after chemotherapy in the preoperative chemotherapy arm. Upon completion of the preoperative chemotherapy treatment, all patients were assessed for their physical conditions before surgery. If patients’ physical conditions did not meet the requirement of surgery, the surgery was delayed until all parameters return to an acceptable level. Surgical resections (lobectomy, bilobectomy, or pneumonectomy) were performed on the basis of the chest CT scan. Mediastinal involvement was assessed by CT. Mediastinal node dissection and sampling were performed by surgeons and pathologists. Patients with pN2 stage NSCLC at surgery received postoperative thoracic radiotherapy. Patients were followed up every 3 months until death or until the cut-off date of this study (May 1, 2012). The median follow-up period for patients in this trial was 53.7998 ± 48.7049 months. BODY.STATISTICAL ANALYSIS: The primary end points of this study included OS and progression-free survival (PFS). OS was defined as the time from the date of randomization to the date of death or May 1, 2012. PFS was calculated from the date of randomization to the time of disease progression, death, or May 1, 2012. The secondary end point was the survival rate associated with clinical remission. The OS and PFS were computed by the Kaplan-Meier method11 and compared by the log-rank test12 and the Cox proportional hazards model.13 The multivariate Cox model13 was used to study variation in the OS and PFS according to major baseline characteristics (age, sex, stage, histology, and treatment). Statistical analyses were conducted using SPSS software, version 13.0 (SPSS, Inc, Chicago, IL, USA). BODY.RESULTS: A total of 356 patients were enrolled in this trial. 179 Patients were then randomly assigned to the preoperative chemotherapy arm, while 177 patients were assigned to the surgery-alone arm (Figure 1). Reasons for ineligibility included stage IIIB tumor (one patient), incomplete resection (eight patients), and missing follow-up (ten patients). Thus, 169 patients in the preoperative chemotherapy arm and 168 patients in the surgery-alone arm were included in the final analysis. The baseline patient characteristics were not statistically different between the two arms (Table 1). BODY.PREOPERATIVE CHEMOTHERAPY: In the preoperative chemotherapy arm, 156 patients completed all 4 cycles, and 13 patients were not evaluable, including six patients who refused to complete chemotherapy. Among the 169 patients who received chemotherapy, the overall chemotherapy response rate was 46.7% (79 patients), and progression of disease was observed in ten patients (5.9%). Chemotherapy-associated adverse events included grade 3 to 4 nausea and vomiting, grade 3 alopecia, grade 3 thrombocytopenia, and grade 3 to 4 neutropenia. After surgery, 83 patients from each arm received adjuvant chemotherapy. BODY.SURGERY: Surgery was performed in 310 patients (92%), including 148 patients (87.6%) in the preoperative chemotherapy arm and 162 patients (96.4%) in the surgery-alone arm. The proportions of patients who underwent lobectomy and pneumonectomy were not significantly different between the two arms (Table 2). A total of 19 patients (6.1%) died after surgery: eight patients (5.4%) in the preoperative chemotherapy arm and eleven patients (6.8%) in the surgery-alone arm. The postoperative deaths were mainly due to a complication from surgery or comorbidities. We did not observe any differences in term of postsurgery recovery between the preoperative chemotherapy and the control arms. BODY.OVERALL SURVIVAL: At the time that this trial ended on May 1, 2012, 261 patients had died, including 128 in the preoperative chemotherapy arm and 133 in the surgery-alone arm. Survival rates at 5, 10, and 15 years were 34%, 28%, and 24%, respectively in the preoperative chemotherapy arm and 48%, 33%, and 21%, respectively in the surgery-alone arm (Figure 2). The median survival time (MST) was 45.42 months in the preoperative chemotherapy arm and 57.59 months in the surgery-alone arm (log-rank P = 0.016), with a HR of death of 1.67 (95% CI, 1.43–1.96). The influences of age, sex, stage, histology, and treatment were assessed by the Cox model. The only relevant factor was disease stage. When the log-rank test was stratified on stage, the survival rate of stage II NSCLC at 5, 10, and 15 years was 27%, 22%, and 22%, respectively in the preoperative chemotherapy arm and 48%, 33%, and 21%, respectively in the primary surgery arm. The MST of stage II NSCLC was 40.86 months in the preoperative chemotherapy arm and 80.81 months in the primary surgery arm (log-rank P = 0.044). There were not significant differences between the two arms in the survival rates of stage I and stage IIIA NSCLC (log-rank P = 0.734; log-rank P = 0.051, respectively). BODY.PROGRESSION-FREE SURVIVAL: No statistical difference in PFS was seen between the two arms, with a HR of progression of 1.135 (95% CI, 0.855–1.506). The median PFS time was 42.21 months in the preoperative chemotherapy arm and 45.67 months in the surgery-alone arm (log-rank P = 0.38). The HR of progression of stage I NSCLC was significantly lower in the preoperative chemotherapy arm when compared with the surgery-alone arm (HR = 0.87; 95% CI, 0.561–1.629; P = 0.027). The risks of progression or death of stage II and stage IIIA NSCLC were not significantly different between the two arms, with HR = 1.226 (95% CI, 0.687–2.186; P = 0.491) for stage II NSCLC and HR = 1.332 (95% CI, 0.877–2.023; P = 0.179) for stage IIIA NSCLC. After the multivariate Cox mode analysis, we found that the relevant factors of disease progression were age (HR = 1.012; 95% CI, 0.996–1.029; P = 0.012) and stage (HR = 1.241; 95% CI, 1.051–1.467; P = 0.011). BODY.SURVIVAL ASSOCIATED WITH REMISSION: Among the 169 patients in the preoperative chemotherapy arm, the clinical remission rate following preoperative chemotherapy was 40.8% (69 patients), including one patient with complete remission and 68 patients with partial remission. The 5-year survival rates were 38.48% and 35.03%, respectively in patients with clinical remission and without clinical remission. The MST was 42.10 months in patients with clinical remission and 35.33 months in patients without clinical remission (log-rank P = 0.630). BODY.SURVIVAL ASSOCIATED WITH CHANGES IN CLINICAL STAGING: Downstaging (clinical stage higher than the pathological stage) was found in 16 patients (9.5%), unchanged staging (clinical stage the same as the pathological stage) in 134 patients (79.3%), and upstaging (clinical stage lower than the pathological stage) in 19 patients (11.2%). There were significant beneficial effects of preoperative chemotherapy in the OS of patients who experienced clinical downstaging compared with the patients who did not experience clinical downstaging. In the downstaging vs unchanged vs upstaging groups, the 5-year survival rates were 44.14% vs 39.52% vs 14.04%, respectively, and the MST was 55.62 months vs 39.52 months vs 25.81 months, respectively (P = 0.03). BODY.DISCUSSION: This long-term follow-up trial demonstrated that preoperative chemotherapy did not produce benefits in the OS for stage I–IIIA NSCLC patients. There was no statistically significant difference in PFS between the two arms. A significant increase in PFS was demonstrated only in the stage I NSCLC patients in the preoperative chemotherapy arm. Due to the inconsistent results from previous clinical trials, the benefit of preoperative chemotherapy in NSCLC has been long debated in the past decade. Similar to the result from our trial, several other trials have also shown that there are not benefits for NSCLC patients in having preoperative chemotherapy. Westeel et al14 reported the long-term results of a French randomized trial that compared preoperative chemotherapy followed by surgery versus surgery alone in resectable (stage I to IIIA) NSCLC (n = 355). After a median 13.8 years of follow-up study, these authors concluded that preoperative chemotherapy did not produce a significant survival benefit, by univariate analysis, but did produce an improved survival in stage I and II, using multivariate analysis.15 The randomized phase III trial reported by Pisters et al16 compared preoperative chemotherapy using paclitaxel and carboplatin followed by surgery, with surgery alone in early-stage NSCLC patients. Although the OS and PFS were higher with preoperative chemotherapy treatment, the differences did not reach statistical significance.16 In the European Organisation for Research and Treatment of Cancer 08012 multicenter randomized trial studied by Gilligan et al,17 519 patients were randomized to receive either surgery alone or 3 cycles of platinum-based chemotherapy followed by surgery. The results of this trial failed to indicate the benefits of preoperative chemotherapy in terms of OS. Consistent with these previous studies, our study also demonstrated that preoperative chemotherapy does not produce significant benefits for NSCLC patients. On the other side, many trials have demonstrated that preoperative chemotherapy is beneficial for NSCLC patients. A trial conducted by Rosell et al8 assigned 60 patients with stage IIIA NSCLC to either have preoperative chemotherapy plus surgery or surgery alone. When an analysis was performed 7 years after patient enrollment, patients in the preoperative chemotherapy arm showed a significant gain in survival. The median survival of the preoperative chemotherapy arm (26 months) was dramatically longer than the surgery-alone arm (8 months) (P < 0.01). Roth et al18,19 conducted a similar trial using chemotherapy, consisting of cyclophosphamide, etoposide, and cisplatin. In that study, the benefit in OS to the preoperative arm was statistically significant (P < 0.008). Meta-analysis of randomized trials has also shown that preoperative chemotherapy improves survival in NSCLC patients. An updated meta-analysis of 13 RCTs, conducted by Song et al,20 showed that preoperative chemotherapy significantly improved the OS of operable NSCLC patients, including patients with stage III NSCLC. However, the benefits of preoperative chemotherapy demonstrated in these trials appear to be modified by multiple factors, such as the number of enrolled patients, the differences between chemotherapy regimens, and the differences between the clinical and pathological stages. The secondary objective of our trial was to assess the survival rate associated with clinical remission induced by preoperative chemotherapy. Although OS was higher with clinical remission after preoperative chemotherapy, the difference was not statistically significant, indicating preoperative chemotherapy was not beneficial to patients even when chemotherapy successfully acted on tumors. There are several factors that may have affected the efficacy of preoperative chemotherapy in our trial. Because patients received at least 1–2 cycles of preoperative mitomycin-cisplatin-vindesine, chemotherapy toxicity may have been responsible for a shortened survival time in the preoperative chemotherapy arm compared with the surgery-alone arm. Furthermore, the mitomycin-cisplatin-vindesine regimen we used in this study is no longer considered to be the first-line chemotherapy regimen for NSCLC. We cannot exclude the possibility that the mitomycin-cisplatin-vindesine regimens may have been a factor that contributed to the negative effect of preoperative chemotherapy in the NSCLC patients. However, recent studies using the same chemotherapy regimen have demonstrated inconsistent results in the benefits of neoadjuvant chemotherapy in NSCLC, suggesting that the choice of chemotherapy regimen may not be a key factor in determining the efficacy of preoperative chemotherapy.10,16 Nevertheless, minimizing chemotherapy-related morbidity and mortality should be given careful consideration when designing future trials, and this may be achieved by choosing less toxic chemotherapy regimens than the one we chose for this trial.21–23 In summary, we found that preoperative chemotherapy had no survival benefits for NSCLC patients with stage I, II, and IIIA diseases. This finding emphasizes the need to develop new strategies to identify the patient subgroups that will be benefited or harmed by preoperative chemotherapy.

TITLE: Sweet taste perception not altered after acute sleep deprivation in healthy young menKeine veränderte Wahrnehmung des Süßgeschmacks bei gesunden jungen Männern durch akuten Schlafmangel ABSTRACT.BACKGROUND: We hypothesized that acutely sleep-deprived participants would rate ascending concentrations of sucrose as more intense and pleasant, than they would do after one night of normal sleep. Such a finding would offer a potential mechanism through which acute sleep loss could promote overeating in humans. ABSTRACT.METHOD: A total of 16 healthy normal-weight men participated in 2 conditions: sleep (permitted between 22:30 and 06:30 h) and total sleep deprivation (TSD) respectively. On the morning after regular sleep and TSD, circulating concentrations of ghrelin and glucose were measured. In addition, participants hunger level was assessed by means of visual analogue scales, both before and after a caloric preload. Finally, following the preload, participants rated both intensity and pleasantness of six orally presented yogurt probes with varying sucrose concentrations (2–29 %). ABSTRACT.RESULTS: Feelings of hunger were significantly more intense under both fasted and sated conditions when subjects were sleep-deprived. In contrast, the change in hunger induced by the preload was similar between the sleep and TSD conditions. Plasma concentrations of ghrelin were significantly higher under conditions of TSD, whereas plasma glucose did not differ between the conditions. No effects were found either on sweet taste intensity or on pleasantness after TSD. ABSTRACT.CONCLUSION: One night of TSD increases morning plasma concentrations of the hunger-promoting hormone ghrelin in healthy young men. In contrast, sweet taste perception was not affected by nocturnal wakefulness. This suggests that an altered sweet taste perception is an unlikely mechanism by which TSD enhances food intake. Short sleep duration is considered a risk factor for weight gain in both children and adults [1]. A breadth of recent evidence suggests that this relation is causal. For instance, experimental sleep loss in healthy young men has been linked to reduced physical activity [2], increased appetite for sweet foods [3, 4], increased circulating ghrelin levels [4, 5], and increased neural responses to images of palatable foods [6]. This latter observation warrants further attention: when viewing food images, sleep-deprived men show enhanced neural activation in brain regions involved in both gustatory and reward processing, including the nucleus accumbens, thalamus, insula, and anterior cingulate cortex. This, in conjunction with previous observations that sleep-deprived men show an increased appetite for sweet foods [3], led us hypothesize that one night of total sleep deprivation (TSD) would enhance perception of sweet taste in healthy young men. To this aim, sweet taste perception was tested in 16 male subjects after either a night of normal sleep or TSD. Previous studies have shown that TSD affects both plasma ghrelin and glucose concentrations [4, 7]. Ghrelin is mainly produced by the stomach and stimulates food intake [8]. In addition, ghrelin has previously been linked to taste perception [9, 10] and food reward evaluation [11, 12] in animal studies. Thus, plasma levels of total ghrelin and glucose, as well as subjective hunger feelings were measured after sleep and TSD respectively. BODY.METHODS: BODY.PARTICIPANTS: A total of 16 healthy male subjects participated in the experiments (age 23 ± 0.9 years; body mass index 23.6 ± 0.6 kg/m2; all non-smokers, with a self-reported regular sleep–wake rhythm [i.e., ~8 h sleep/night] during the 6 weeks before the experiments, and not on any medication). Sleep disturbances during the intervention were excluded by electroencephalography (EEG) sleep monitoring. The design included an adaptation day including an overnight sleep that served to habituate participants to the experimental setting. The study was approved by the Regional Ethical Review Board in Uppsala, and the procedures followed were in accordance with the Helsinki Declaration. All participants gave written informed consent and were paid for their participation in the study. BODY.STUDY DESIGN AND PROCEDURE: In a randomized and balanced crossover design, each subject participated in two conditions: 8-hours of sleep opportunity (‘sleep’) and total sleep deprivation (TSD). Upon arrival, participants started their adaptation day, which comprised a night of sleep followed by a standardized day in which they received three regular meals and one snack in fixed amounts, and two 30-min walks. This day was followed by a nighttime intervention period (22:30–06:30 h) in which subjects slept or stayed awake, after which they conducted the sweet intensity ratings as described below. Blood was sampled at 07:30 h after the nighttime intervention day. In the sleep condition, lights were turned off at 22:30 h, and switched on the next morning at 06:30 h. Polysomnography was performed using Embla A10 recorders (Flaga hf, Reykjavik, Iceland) and comprised EEG, electrooculography (EOG), and electromyography (EMG). Sleep stages were determined according to standard criteria [13] by an experienced scorer blinded to the study hypothesis. To keep subjects awake in the TSD condition, they were allowed to spend their time with a selection of movies, games, and books, they had access to bottled water, and lights were kept on. Participants were continuously monitored by the experimenters. In the morning after regular sleep or nocturnal wakefulness, subjects were requested to rate their appetite sensations (hunger, fullness, desire to eat, prospective consumption) and thirst on a 100-mm visual analog scale (VAS) at 07:00 h. A blood sample was taken at 07:30 h for hormonal measurements, after which participants again rated their appetite sensations at 08:00 h. To minimize the potential confound of hunger evoked by TSD, immediately after these ratings, they received a caloric preload consisting of 2 packages (500 ml in total; 100 kcal/100 g) of Gainomax Recovery Vanilj (Norrmejerier Ek. För., Umeå, Sweden) poured into 2 glasses, and 2 bars (75 kcal/bar) of a Wasa Sandwich Cream Cheese Naturell (Barilla Sverige AB, Stockholm, Sweden), providing 650 kcal. This preload was consumed in its entirety within 10 min. Following the preload, participants again reported appetite ratings (08:30 h), and conducted the sweet taste intensity rating task (09:00 h). BODY.SWEET TASTE INTENSITY: A standard food (yogurt; Mild Lättyoghurt Naturell, Arla Foods, Viby, Sweden) was presented to which sucrose was added at different concentrations: 2 %, 5 %, 9 %, 15 %, and 22 % and 29 % (w/w; i.e., gram sucrose per 100 ml yoghurt). Participants tasted a single spoonful and rated sweetness intensity and hedonic value (pleasantness) of the different sucrose concentrations on a 100-mm VAS, anchored with the terms “not at all” and “extremely”. Participants were then asked to return the remaining yogurt and to neutralize their taste by eating plain crackers and drinking water. This procedure was repeated for all six sucrose concentrations. The order of presentation was randomized within participants. BODY.BIOCHEMICAL ANALYSIS: Blood samples were centrifuged immediately after sampling. The supernatant was stored at −80 °C, for analysis of plasma ghrelin and glucose. Concentrations of total ghrelin were assessed using commercially available ELISA kits for humans (EZGRT-89 K; Millipore, Billerica, MA, USA). Plasma glucose was measured using routine assays (hexokinase method, Aeroset; Abbott Diagnostics, North Chicago, IL, USA). BODY.DATA ANALYSIS: Data were analyzed using SAS (version 9.5; SAS Institute Inc.), and are presented as means (± SEM) unless otherwise indicated. The effect of sleep deprivation on ratings of sweetness intensity and pleasantness was tested using repeated ANOVA measures (within-subject factors: Sleep/TSD, Sucrose concentration). In order to check that intra-individual differences did not account for nonsignificant effects in the small sample size, we repeated these analyses using normalized values. Since TSD was hypothesized to increase plasma ghrelin and feelings of hunger, a one-tailed p value of < 0.05 was considered significant. In case of the sweet perception task, a two-tailed p value < 0.05 was considered significant. BODY.RESULTS: BODY.APPETITE SENSATIONS: Hunger ratings on the adaptation day did not differ between the sleep and TSD conditions (data not shown). In the morning after nocturnal wakefulness, participants reported greater hunger than they did after normal sleep (TSD vs. Sleep, 76 ± 7 vs. 61 ± 6 mm, p = 0.01 at 07:00 h; 80 ± 4 vs. 69 ± 6 mm, p = 0.08 at 08:00 h; 43 ± 7 vs. 33 ± 7 mm, p = 0.04 at 08:30 h [i.e., 20 min after the preload]). However, no significant differences between the TSD and sleep conditions were observed when the change in hunger before (i.e., 08:00 h) and after the preload (i.e., 08:30 h) was analyzed (TSD vs. Sleep, −37 ± 6 vs. −36 ± 5 mm). Other appetite ratings (fullness, desire to eat, and prospective consumption) yielded similar results, with corresponding changes following the preload (data not shown). BODY.SWEET TASTE PERCEPTION AND PLEASANTNESS: Ratings of sweet intensity or pleasantness of the yogurt samples with varying sucrose concentrations did not differ between conditions (Fig. 1). Similar null results were obtained when ratings of the sweet taste perception task were normalized (Fig. 1). Fig. 1Mean (± SEM) absolute and normalized ratings of sweet taste intensity and pleasantness of yogurts with increasing sucrose concentrations: 2 %, 5 %, 9 %, 15 %, 22 %, and 29 % (i.e., gram sucrose per 100 ml yoghurt, % [w/w]) after both sleep (dashed line) and total sleep deprivation (solid line) conditions. As indicated by repeated measures ANOVA, ratings of sweet intensity or pleasantness of the yogurt samples with varying sucrose concentrations did not significantly (i.e., p < 0.05) differ between conditions BODY.SLEEP RECORDINGS: Sleep in the sleep condition was typical for laboratory conditions (Tab. 1). The sleep-onset latency was 8 ± 4 min, and sleep efficiency was 92 ± 1 %. Tab. 1Polysomnographic characteristics (mean ± SEM) of the second night in the sleep condition Parameter Time in bed (min) 480 Sleep onset latency 8 ± 4 After sleep onset     Total sleep time (min) 442 ± 6     Wake (min; B) 30 ± 3     Sleep stage 1 (min) 5 ± 1     Sleep stage 2 (min) 219 ± 11     Slow wave sleep (min) 115 ± 6     Rapid eye movement (REM) sleep (min) 103 ± 8 Sleep efficiency (%)a 92 ± 1 aSleep efficiency = total sleep time/time in bed. BODY.GLUCOSE AND HORMONAL MEASUREMENTS: Plasma glucose measurements did not differ between the TSD and sleep conditions (TSD vs Sleep, 5.4 ± 0.5 vs. 5.3 ± 0.4 mmol/l). Plasma concentrations of total ghrelin were higher after TSD than after a night of sleep (TSD vs. Sleep, 442 ± 61 vs. 390 ± 44 pg/ml, p = 0.04). BODY.DISCUSSION: Here we show that one night of wakefulness (which is common in shift workers) increases morning plasma concentrations of the hunger-promoting hormone ghrelin in healthy young men. In contrast, perception of sweet taste tested in a broad range of sucrose concentrations was not affected. This suggests that altered processing of sweet tastes is an unlikely the mechanism by which one night of total sleep deprivation (TSD) affects the homeostatic and hedonic control of eating in humans [6, 14, 15, 16, 17]. It has been demonstrated that humans who receive an intravenous bolus of the hormone ghrelin while viewing images of palatable food show an increased activity in brain areas intimately linked to gustatory and reward processing (e.g., amygdala, orbitofrontal cortex, anterior insula, and striatum) [18]. In line with these observations, high circulating levels of ghrelin have been linked to enhanced consumption and preference for sweet tasting food in animals [9]. Against this background, we hypothesized that acute sleep deprivation—a condition that causes an increase in morning plasma concentrations of ghrelin, as shown here and by others [5, 19]—would modulate the perception of sweet taste in healthy young men. Such a finding would offer a potential mechanism through which sleep loss may promote overeating in humans. However, in contrast to our hypothesis, a night of wakefulness affected neither taste intensity nor pleasantness ratings when subjects were administered six ascending concentrations of sucrose in yogurt samples. There are a variety of explanations for why we did not observe the predicted effects. A range of metabolic, physiological, and genetic variables that vary from person to person are known to moderate the ability to perceive sweetness [20]. With this in mind, we cannot rule out the possibility that the potential effects of TSD on sweet perception were masked by inter-individual differences in sweet taste perception resulting from differences in these variables [21]. However, normalizing the results of the sweet taste perception task to account for the ability to perceive sweetness failed to produce significance. Another possibility is that the caloric preload reduced the sensitivity of the test, and that participants in the fasted state would have shown significant differences between conditions. Additionally, caution should be exerted when extrapolating our results, as the experiment focused exclusively on sweetness, at the exclusion of other potentially significant tastes such as salt, bitter, sour, and umami. Sleep loss has been shown to increase the responsively to stress in humans [22]. As stress can cause a generalized decrease in sensitivity to rewards (i.e., anhedonia) [23], our negative results do not exclude that acute sleep deprivation in conjunction with stress may alter the way humans perceive sweet taste. Finally, whether sweet taste perception remains also stable under periods of chronic partial sleep deprivation, or whether our results generalize to females or other age groups, requires further investigation. BODY.CONCLUSION: Our data add further evidence that acute sleep loss increases circulating concentrations of the hunger-promoting hormone ghrelin in healthy young men [4, 5]. In contrast, TSD did not produce differences in sweet taste perception. This suggests that the increase in daytime food intake after nocturnal sleep deprivation may be caused by endocrine mechanisms independently from the subjective perception of sweetness.

TITLE: Comparative Study with a Lip Balm Containing 0.5% Propolis Special Extract GH 2002 versus 5% Aciclovir Cream in Patients with Herpes Labialis in the Papular/Erythematous Stage: A Single-blind, Randomized, Two-arm Study ABSTRACT.BACKGROUND AND OBJECTIVES: This controlled single-blind trial compared the efficacy of a lip balm with propolis special extract GH 2002 at a concentration of 0.5% in the treatment of episodes of herpes labialis with that of 5% aciclovir cream. ABSTRACT.METHODS: Patients in the erythematous/papular stage were randomized: 189 patients were treated with propolis cream, 190 patients were treated with aciclovir cream (intention-to-treat population). Application was 5 times daily. The primary parameter was the difference in median time to complete encrustation or epithelialization of lesions. Secondary parameters were the development of typical herpes symptoms (eg, pain, burning and itching, tension, and swelling), the global assessment of efficacy, and the safety of application. ABSTRACT.RESULTS: The predefined clinical situation was reached after a median of 4 days with propolis and after 5 days with aciclovir (P < 0.0001). Significant differences in favor of the study preparation were found with all secondary parameters and symptoms. No allergic reactions, local irritations, or other adverse events were observed. ABSTRACT.CONCLUSIONS: A formulation of 0.5% propolis GH 2002 extract lip balm was found to be superior in the treatment of episodes of herpes labialis over 5% aciclovir cream in patients in the papular/erythematous phase upon inclusion. EudraCT Registration No. 2006-001971-38. BODY.INTRODUCTION: Episodes of herpes labialis typically start with a prodromal phase with local pain, tingling, and burning followed by erythematous and papular phases with inflamed and reddened papules, followed by a vesicular phase with fluid-filled blisters. Via the ulceration phase or the bursting of the vesicle with wound formation, it finally leads to the incrustation and healing phase.1 The typical duration of the natural course of untreated herpes labialis is 7 to 10 days and sometimes up to 15 days.1, 2 Accepted local and systemic standard treatments for herpes labialis consist of nucleoside analogues of the aciclovir type.3, 4, 5 Recently, a number of studies demonstrated antiviral effects of propolis special extract GH 2002 against herpes simplex virus type I and II in vitro.6, 7 In addition, antimicrobial effects of GH 2002 were demonstrated in vitro against methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium, Candida, and Streptococcus pyogenes.8 A clinical dose-finding study compared concentrations of 0.1%, 0.5%, and 1% of propolis extract GH 2002 in a lip balm for the treatment of herpes labialis.9 The study resulted in the recommendation of 0.5% as a concentration with a sufficiently strong effect and an acceptable level of safety. The latter point was also examined and confirmed in an open observational study.10 The intention of the present study was the evaluation of the clinical usefulness and formal demonstration of efficacy of 0.5% GH 2002 in a lip balm through a reference-controlled trial, using a cream containing 5% aciclovir as an accepted topical comparator medication with marketing authorization for the treatment of herpes labialis. BODY.MATERIALS AND METHODS: The study was designed as a randomized single-blind, parallel group, reference-controlled multicenter trial (clinical Phase III), with a comparison of the effects of topically applied 0.5% propolis GH 2002 extract lip balm and 5% aciclovir cream in the treatment of herpes labialis. It was performed at the dermatology department of the Medicinal Faculty of the Charles University of Prague and at 3 dermatology ambulatory care centers with participants recruited from regular outpatients presenting for herpes episodes. No financial incentive was given for study participation. Patients were assigned a consecutive random number according to their entry into the trial. A random list in blocks of 10 ensuring a balanced distribution of patients to the 2 study arms was prepared in advance by the study sponsor using a random number generator (RandList version 1.2; DatInf GmbH, Tübingen, Germany). The random list was closed after labeling and only reopened after official closing of the study and the database during analysis. The physicians had no access to the random code. The study duration was up to 10 days or up to the visit when the lesions were fully epithelialized or encrusted. Examinations and documentations by the physician were made at Days 0, 2, 3, 4, and 5. Additional examinations on Days 8 (±1 day) and 10 were foreseen for patients still requiring therapy at the previous visit. BODY.STUDY MEDICATION: One study arm received a lip balm containing 0.5% propolis special extract GH 2002 (Herpetino batch No. 20/0913; Gehrlicher Pharmazeutische Extrakte, Eurasburg, Germany). The active constituent of the study preparation was the purified propolis semiliquid extract GH 2002 (extract batch No. 9494, drug extract ratio 2:1, extraction solvent ethanol; excipients in the lip balm: water, xylitol, Butyrospermum parkii, hydrogenated polyisobutene, Simmondsia chinensis seed oil, Prunus amygdalis dulcis (sweet almond) oil, panthenol, polyglyceryl-4-isostearate, cetyl PEG/PPG-10/1, dimethicone, hexyl laurate, sodium chloride, tocopheryl acetate, and bisabolol). The extract GH 2002 is purified from potentially allergenic pollen, waxes, and resins, and is standardized to a defined content of flavonoids, polyphenols, and phenylcarboxylic acids.11 The second study arm applied a 5% aciclovir cream (Herpesin batch No. 10/1013; Teva Pharmaceuticals CR, Prague, Czech Republic) with ingredients including carbomer 934, sodium hydroxide, dimethicone, cetyl alcohol, sodium lauryl sulphate, methyl parabene, and purified water. The 2 creams were manufactured to be similar in appearance and consistency; both were prepared as externally undistinguishable tubes with 10 g cream (sufficient for a study period of 10 days) and were delivered to the study center with a prenumbered code according to the blinded random number list. The content of the tubes differed slightly with respect to color and odor, which is why the study was considered single-blinded because as the physicians might have known the color and odor difference. The patients were not expected to know the difference between the study preparations; thus, no undue bias was expected. Each preparation was applied 5 times daily (every 3–5 hours) to the entire upper and lower lip, corresponding to a daily dose of approximately 1 g cream. BODY.PRIMARY AND SECONDARY PARAMETERS: The first aim of the study was the comparison of median times to full encrustation or epithelialization between groups. According to previous experience with aciclovir and propolis extract the median time to this clinical point was estimated to be reached within approximately 5 days. Treatment was planned for up to 10 days to cover the typical duration of an untreated episode of herpes labialis. After 10 days the healing process should be in its final stages even in patients not responding to the study medication. Secondary parameters were the assessments of typical symptoms of lip sores (pain on a visual analogue scale, itching/burning, and tension/swelling on a 4-point verbal rating scale), and an evaluation of global efficacy by the physician on a 4-point verbal rating scale. A descriptive analysis of the development of the individual symptoms of the episode was foreseen as a secondary outcome parameter, including the percentage of patients skipping the vesicular or erosive phase and the calculation of the number needed to treat. Subgroup analyses according to age and gender were also planned. Safety of application was addressed through an analysis of adverse events, including allergic reactions and skin irritation actively discussed with the patients at each visit. In addition, patients were asked to bring the medication tubes back for inspection for the assessment of compliance. BODY.INCLUSION AND EXCLUSION CRITERIA: Patients of both genders aged 18 to 70 years could only be included if they had visible eruptions (erythematous or papular) for no more than 30 hours before the first examination, and if their history showed at least 4 previous episodes of herpes labialis. Patients in the prodromal stage; that is, with burning or tension of the lips only, and patients with progressed stages (ie, vesicular, erosive, or encrusted) were not eligible. The inclusion and exclusion criteria with respect to the stage of the episode had to be strictly adhered to because they were the foundation of a comparability of results with published data on aciclovir. Further exclusion criteria were hypersensitivity to any component of the test preparation or the reference, concomitant viral infections, acquired or malignant immunodeficiency, including HIV or leukemia, the severity of the herpes labialis requiring systemic treatment, or the concurrent use of other topical preparations or systemic antiviral medication. The use of such preparations was also not permitted for the duration of the trial. BODY.CASE NUMBER CALCULATION: A case number calculation was made based on the results of a previously performed dose-finding study.9 The study preparation had reduced the time to complete encrusting of herpes lesions by approximately 1 day compared to a 0.1% formulation, which still showed activity. It was therefore anticipated that 0.5% propolis would reduce time to complete encrusting or epithelialization by approximately 1.5 days compared with the untreated course of the episode. Aciclovir shortens the duration of herpes labialis episodes by approximately 1 day versus placebo. The case number calculation was therefore based on the assumption that the study preparation should be superior over aciclovir by approximately 0.5 days, as judged by the median time to full encrusting or epithelialization. The case number calculation was based on an expected dropout rate of 20% and a power of at least 80% in the superiority testing, which led to the conclusion of 190 patients to be included per group. BODY.VOTE OF THE ETHICS COMMITTEE: The study was planned and carried out in accordance with the criteria of Good Clinical Practice and the ethical standards defined in the Declaration of Helsinki. An approval of the ethics committees of the study centers (Etická komise fakultní nemocnice Královské Vinohrady, Srobárova 50, 10035 Praha 10; and Etická komie pro multicentrické klinické hodnoceni fakultní nemocnice v Motole, V uvalu 84, 15005 Praha 5) and the Czech drug authorization authority, as well as a signed informed consent form for all participants was obtained. Initiation of the trial was on February 24, 2012. The final visit of the last participant took place on January 14, 2014. BODY.STATISTICAL ANALYSIS: IBM-SPSS Statistics version 21.0.0 (IBM-SPSS Inc, Armonk, NY) was used as the statistical software to perform the analyses. The intention-to-treat (ITT) group included all patients who received study drugs and returned for at least 1 visit after the admission examination. The per-protocol group (PP) included all patients who followed the protocol. The safety population comprised all patients exposed to study medication. Superiority calculation was made primarily in the ITT population through a 1-sided Mann-Whitney U test by the SPSS Exact module giving exact P values, with a level of significance set to 0.025. The threshold for superiority was defined as a difference between medians of the groups for full encrustation or epithelialization with minimum of 0.5 days in favor of propolis. Missing values were to be replaced by the worst-case imputation method, replacing missing values in the propolis group by the worst value of the propolis group, and missing values in the aciclovir group by the best value of the aciclovir group. The worst-case imputation is favorable for the comparator and makes superiority testing more robust. Individual improvement of pain as a secondary parameter was the difference of the average pain at Visits 3 and 4 minus pain at Visit 1. Between-group comparisons were made with the group averages using the Mann-Whitney U test at a 2-sided significance level of 0.05. Presence and intensity of itching/burning and tension/swelling, and the physician’s global assessment of efficacy were compared between groups using the Fisher exact test and Mann-Whitney U test at a 2-sided significance level of 0.05. All other parameters were analyzed descriptively, with significance between groups examined using the Mann-Whitney U test or the Fisher exact test, as appropriate. All P values for secondary end points do not possess confirmatory value. BODY.RESULTS: BODY.DEMOGRAPHIC DATA: Three hundred seventy-nine outpatients with the diagnosis of herpes labialis were included (ITT population) (Figure 1). The inclusion and exclusion criteria, including those specifically referring to excluded comedication, were met in all cases. Two hundred thirty-seven patients were women and 142 were men. One hundred eighty-nine patients were assigned to the study preparation (mean (SD) age 40.7 [13.0] years and 58.2% female), 190 to reference (age 41.1 [13.8] years and 66.8% female). There were no statistically significant differences between groups with respect to age and expression of herpes lesions.Figure 1Flow chart of patient distribution to treatment groups. ITT = intention-to-treat population; PP = per protocol population. Fig. 1 Four patients prematurely discontinued participation in the study: 1 patient with propolis (lost to follow-up) and 3 patients with aciclovir (1 patient lost to follow-up and 2 patients for lack of efficacy). The PP population therefore consisted of a total of 375 participants (188 treated with the study preparation and 187 treated with the reference preparation). BODY.DEVELOPMENT OF LESIONS OVER TIME: As demanded by the inclusion criteria, all patients presented only erythematous or papular lesions at inclusion. The further development shows distinct and statistically significant differences between groups: at Day 4, 55.0% of the participants treated with propolis had no more erythema, papules, vesicles, or erosions (Figure 2). This increased to 89.9% on Day 5. With aciclovir, the same clinical end point was reached for only 25.3% of patients, whereas on Day 5, 57.5% of patients were fully encrusted or epithelialized (Figure 3). In addition, significantly fewer study participants treated with the study preparation than patients treated with reference preparation developed vesicles or erosions (42% vs 85% vesicles and 39% vs 72% erosions; in both cases P < 0.0001 by Fisher exact test). Converting these figures into numbers needed to treat, the comparison results in 2.3 and 3.0 patients treated with reference to avoid the formation of vesicles and erosions to the same extent as for 1 patient treated with the study preparation.Figure 2Development of herpes lesions treated by propolis extract. (Intention-to-treat population n = 189). Fig. 2Figure 3Development of herpes lesions treated by aciclovir (intention-to-treat population; n = 190). Fig. 3 BODY.SUPERIORITY TESTING: The course of the herpes episodes, as shown in Figure 2, Figure 3, gives a first impression on the clinical effects of propolis treatment versus aciclovir. For statistical comparison in the context of superiority testing, the course of the herpes episodes had to be transformed into statistically comparable figures. The mathematical approach was based on the median time to reach full encrustation or epithelialization in the population of each study arm. The superiority test was performed as planned in the ITT population, with a threshold of a difference between groups of 0.5 days in favor of propolis. The median time to complete encrustation/epithelialization was 4.0 days with propolis (average 4.41 [1.63] days), and 5.0 days with aciclovir (average 5.54 [1.87]). The superiority of propolis was highly significant (P < 0.0001; Mann-Whitney U test modified for superiority testing with a threshold 0.5 days; Figure 4).Figure 4Superiority testing: Time in days to complete encrustation or epithelialization of herpes lesions (intention-to-treat population n = 189 for propolis and 190 for aciclovir). Bars represent the 25th to 75th percentile. Fig. 4 BODY.DEVELOPMENT OF PAIN: Mean (SD) pain at baseline, assessed on a 100-mm visual analogue scale, was 34 (25) with propolis and 39 (26) with aciclovir (ITT). Pain decreased continuously in both groups during the study, but the difference between means was 10 mm on a visual analogue scale and more at the visits on Days 2, 3, and 4 (Figure 5). The predefined calculation of the difference of the average of pain at days 3 and 4 to pain at baseline (V1) resulted in a pain reduction of 27 mm visual analogue scale with the propolis and 19 mm with aciclovir. The difference of 8 mm on a visual analogue scale in favor of propolis extract GH 2002 was statistically significant (P < 0.001 based on Mann-Whitney U test).Figure 5Development of pain assessment by visual analogue scale (VAS) (intention-to-treat population). Fig. 5 BODY.ITCHING/BURNING AND TENSION/SWELLING: Itching/burning and tension/swelling were evaluated through a 4-step verbal rating scale (absent, mild, moderate, and severe). At baseline, approximately 30% of patients indicated the absence of itching or burning and approximately 10% the absence of tension or swelling, with no statistically significant difference between groups (ITT population). Overall, patients in the aciclovir group had more severe tension and swelling than patients in the group using propolis (P = 0.042 based on Mann-Whitney U test; data not shown). For the further visits on Days 2 to 5 there was a significant difference in favor of propolis, reaching 89% versus 66% free of itching or burning (Figure 6), and 94% versus 74% free of tension and swelling (Figure 7) at Day 5 for propolis versus aciclovir (P < 0.001 based on Mann-Whitney U test). Results obtained with the PP population were practically identical.Figure 6Percentage of patients free of burning and itching during the course of the study (intention-to-treat population). Fig. 6Figure 7Percentage of patients free of tension and swelling during the course of the study (intention-to-treat population). Fig. 7 BODY.GLOBAL ASSESSMENT OF EFFICACY BY A PHYSICIAN: Differences between groups in favor of propolis were also confirmed in the assessment of global efficacy by the physicians (Figure 8). At the visit on Day 5, 94.7% of patients treated with propolis and 68.4% of patients treated with aciclovir received efficacy assessments of “good” to “very good.” Differences were statistically significant at all visits on Days 2, 3, 4, and 5 (ITT group P < 0.0001 based on Mann-Whitney U test). Results for the PP group were practically identical, with 94.7% and 69.0% of ratings as “good” and “very good” on Day 5 for propolis and aciclovir, respectively.Figure 8Global assessment of efficacy by the physician (intention-to-treat population). Fig. 8 BODY.SUBGROUP ANALYSES: Subgroup analyses according to age and gender indicate that young female patients may have profited to a slightly larger extent. The difference of means with respect to the difference in time to full encrustation/epithelialization was 1.2 days for women, and 0.95 days for men. Similarly, younger patients profited slightly more than older patients (1.4 vs 0.8 days). The strength of the effect of propolis and aciclovir was not influenced by the severity of symptoms at baseline. BODY.SAFETY AND COMPLIANCE: In both treatment groups there were no adverse events observed, which includes the absence of local reactions or superinfections. This finding is in line with the experience with the application of both study preparations. There was no hint of poor compliance through the safety assessment and the inspection of the returned medication for consumption. BODY.DISCUSSION: The preparation 0.5% propolis GH 2002 lip balm tested in this study has previously shown positive results in the treatment of herpes labialis,9, 10 but has not yet been tested against an active reference in a controlled study. For a better comparability of outcomes, the design and end points of this study were selected to match those of placebo-controlled studies with aciclovir, and to justify the use of aciclovir as a reference treatment for the verification of the therapeutic applicability of the propolis preparation. GH 2002 cream with 0.5% propolis special extract was found to be superior to 5% aciclovir cream under the clinical conditions defined in this study; that is, a start of treatment in the early, erythematous or papular phase. The advantage of propolis lip balm over aciclovir was especially visible within the first 3 to 5 days after the beginning of treatment. It is no contradiction that after 10 days there was no longer a significant difference between propolis and aciclovir lip balm: episodes of herpes labialis are self-limiting conditions and would normally be expected to heal within 10 to 14 days, if left untreated. The aim of a medication is to shorten this period, and a shortening of the herpes episode was demonstrated for both aciclovir and propolis extract. The lack of a placebo control might be considered a downside of the study protocol. However, the efficacy of aciclovir in the tested concentration is generally accepted as proven, and can also be derived from the outcome of this study. In the Czech Republic, where this study was performed, the use of placebo is considered ethically unacceptable when there is a reference treatment with accepted efficacy. The dosing of aciclovir corresponded to a dose accepted as efficacious. The worst-case imputation method for missing values would be in favor of aciclovir. The median and average time to complete encrustation or epithelialization was approximately 1 day shorter in the propolis group than in the aciclovir group, which underlines the clinical importance of the findings beyond statistical significance. In addition, a higher proportion of patients in the propolis group skipped the vesicular and the erosive stage. The measured advantage of the study preparation over the reference population with respect to pain reduction, reaching 8 mm on the VAS at mid study, was considered of borderline clinical importance. Such a difference is still noticeable to the patients,12, 13 especially because the patients treated with propolis reached a defined amount of pain improvement approximately 1 to 2 days earlier than patients treated with the reference balm. BODY.CONCLUSIONS: An important aspect of studies in the treatment of herpes labialis is the selection of a clinically exact defined starting point for the assurance of comparability between groups. In this study, all patients were in the papular or erythematous stage. The findings are therefore applicable to the early stages of a herpes episode. We are already examining the question of whether the advantages of propolis over aciclovir are still visible if lip balm is first applied when patients are already in the vesicular stage. BODY.CONFLICTS OF INTEREST: This clinical study was supported by funds from the companies Harras Pharma Curarina GmbH (Munich, Germany; applicant for the marketing authorization of the study preparation) and Gehrlicher Pharmazeutische Extrakte GmbH (Eurasburg, Germany; manufacturer of the study preparation). The study sponsors proposed the trial and provided funding and honoraria for the data collection, the statistical evaluation, and manuscript preparation. The sponsors were not involved in the planning, execution, data analysis and evaluation, and the publication of the clinical trial results. These tasks were carried out under the responsibility of the principal investigator.

TITLE: Lack of the effect of lobeglitazone, a peroxisome proliferator-activated receptor-γ agonist, on the pharmacokinetics and pharmacodynamics of warfarin ABSTRACT.AIMS: Lobeglitazone has been developed for the treatment of type 2 diabetes mellitus. This study was conducted to evaluate potential drug–drug interactions between lobeglitazone and warfarin, an anticoagulant with a narrow therapeutic index. ABSTRACT.METHODS: In this open-label, three-treatment, crossover study, 24 healthy male subjects were administered lobeglitazone (0.5 mg) for 1–12 days with warfarin (25 mg) on day 5 in one period. After a washout interval, subjects were administered warfarin (25 mg) alone in the other period. Pharmacokinetics of R- and S-warfarin and lobeglitazone, as well as pharmacodynamics of warfarin, as measured by international normalized ratio (INR) and factor VII activity, were assessed. ABSTRACT.RESULTS: The geometric mean ratios (GMRs) and 90% confidence intervals (CIs) for area under the curve from time zero to the time of the last quantifiable concentration (AUClast) for warfarin + lobeglitazone: warfarin alone were 1.0076 (90% CI: 0.9771, 1.0391) for R-warfarin and 0.9880 (90% CI: 0.9537, 1.0235) for S-warfarin. The maximum observed plasma concentration (Cmax) values were 1.0167 (90% CI: 0.9507, 1.0872) for R-warfarin and 1.0028 (90% CI: 0.9518, 1.0992) for S-warfarin, both of which were contained in the interval 0.80–1.25. Lobeglitazone had no effect on the area under the effect–time curve from time 0 to 168 hours (AUEC) of INR and factor VII activity, as demonstrated by the GMRs of 1.0091 (90% CI: 0.9872, 1.0314) and 0.9355 (90% CI: 0.9028, 0.9695), respectively. In addition, the pharmacokinetics of lobeglitazone was also unaffected by warfarin. ABSTRACT.CONCLUSION: Concomitant administration of lobeglitazone and warfarin was well tolerated. Lobeglitazone had no meaningful effect on the pharmacokinetics or pharmacodynamics of warfarin. These findings indicate that lobeglitazone and warfarin can be coadministered without dosage adjustments for either drug. BODY.INTRODUCTION: Thiazolidinediones (TZDs) are synthetic ligands for peroxisome proliferator-activated receptor-gamma (PPAR-γ), which is expressed primarily in adipocytes and to a lesser extent in muscles. PPAR-γ activation stimulates fatty acid storage in adipocytes, which decreases the availability of free fatty acids and adipocyte-derived signaling molecules and improves insulin sensitivity in skeletal muscle.1 Lobeglitazone (CKD-501; Chong Kun Dang Pharmaceutical Corp, Seoul, Korea) is a PPAR-γ agonist with substituted pyrimidine derivatives containing TZD.2 Lobeglitazone has been shown to have more potent activity than the prototypic TZDs (ie, pioglitazone and rosiglitazone) in both in vitro and in vivo studies.2,3 In healthy volunteers, lobeglitazone has a favorable tolerability profile and exhibits linear pharmacokinetics (PK) over the dose range of 0.5–4.0 mg once daily.4 Following oral administration, lobeglitazone is rapidly absorbed with a time to maximum plasma concentration (Tmax) of 1.0–3.0 hours and is eliminated with a mean elimination half-life (t1/2) of 7.8–9.8 hours.1 On the basis of the favorable balance in the efficacy and safety profile, lobeglitazone was approved for the treatment of diabetes in 2013 by the Ministry of Food and Drug Safety, Republic of Korea.5 The use of warfarin, the mainstay of oral anticoagulant therapy, is often complicated by the narrow therapeutic index and wide interindividual variability. The dosage and administration of warfarin should be individualized for each patient according to the patient’s international normalized ratio (INR) response to the drug. Furthermore, small changes in its PK may lead to the need for dose adjustment. Patients with diabetes are at increased risk of thromboembolic events.6 It has been estimated that higher blood glucose levels contribute to an additional 1.5 million deaths due to ischemic heart disease and 0.7 million deaths due to stroke every year.7 Patients with type 2 diabetes mellitus at risk of thromboembolic events are likely to be prescribed anticoagulation therapy, and the vitamin K antagonist warfarin is commonly used for this purpose.8 Hence, it is important to determine whether there are drug–drug interactions between warfarin and antidiabetic drugs. In vitro studies have indicated that lobeglitazone and its metabolites are unlikely to interfere with the PK of warfarin. However, because of the narrow therapeutic index of warfarin, the current clinical study was conducted to confirm the preclinical results that suggested a lack of clinical drug–drug interaction between lobeglitazone and warfarin. We believe that this information is needed because of the safety concerns associated with many TZDs9,10 and because case reports have identified interactions with warfarin.11,12 To this end, the present study (ClinicalTrials.gov registry number: NCT02002611) assessed PK, pharmacodynamics (PD), and safety of warfarin and lobeglitazone when administered alone and concomitantly in healthy subjects. BODY.MATERIALS AND METHODS: BODY.SUBJECTS: Eligible subjects were healthy males aged between 19 years and 55 years who had a body mass index within the range of 19–27 kg m−2. To be included, each subject had to have a clinically acceptable 12-lead electrocardiogram (ECG), vital signs, and physical examination results. Key exclusion criteria for the study included the following: 1) a history of hemorrhagic disease or bleeding tendency; 2) INR or activated partial thromboplastin time outside of the normal range; 3) within 30 days before screening, use of any medication that could affect the results of the study; 4) a history of hypersensitivity or allergic reaction to any of the study drugs; 5) a history of hereditary problems, such as galactose intolerance, Lapp lactase deficiency, or glucose–galactose malabsorption. BODY.STUDY DESIGN: This was a single-center, open-label, randomized, two-sequence, two-period crossover study. One period (treatment AB) comprised treatment A, a once-daily oral dose of lobeglitazone (0.5 mg) for 4 days (days 1–4), and treatment B, a single oral dose of warfarin (25 mg) on day 5 and a single oral dose of lobeglitazone (0.5 mg) for 8 days (days 5–12). In the other period (treatment C), a single oral dose of warfarin (25 mg) was administered. Subjects received one of two dosing schedules: AB, followed by C, or C, followed by AB with a ≥10-day washout interval. The doses for all treatment periods were administered with 240 ml of water in the morning following an overnight fast. Blood samples were obtained for characterization of the PK and PD and were collected at the predose and selected time points. The study (Clinical trial registration number: NCT02002611) was conducted according to the ethical principles of the Declaration of Helsinki. The study protocol and any amendments were reviewed by the Institutional Review Board of Samsung Medical Center, Seoul, Republic of Korea. Written informed consent was obtained from each subject before enrollment in the study. BODY.PK ASSESSMENT: Lobeglitazone concentration was analyzed using a validated liquid chromatography procedure (Shimadzu UFLC; Shimadzu, Kyoto, Japan) and detected by tandem mass spectroscopy (5500 Qtrap; AB Sciex, Framingham, MA, USA) in the positive ionization mode. Pioglitazone was used as the internal standard, and the lower limit of quantification for lobeglitazone was 0.05 μg L−1. The coefficients of variation (CVs) for between-run and within-run variability were ≤6.88% and ≤8.29%, respectively, with mean deviations from the nominal concentration of no more than ±4.93%. R- and S-warfarin concentrations were analyzed using a validated liquid chromatography method (Shiseido Nanospace SI-2; Shiseido, Tokyo, Japan) and detected by tandem mass spectroscopy (API 4000; AB Sciex) in the negative ionization mode. The lower limit of quantification for R- and S-warfarin was 2.5 μg L−1. The CVs for between-run variability were ≤3.14% for S-warfarin and ≤3.95% for R-warfarin. The between-run accuracy, expressed as mean deviation from the nominal concentration, was no more than ±6.04% for S-warfarin and ±4.48% for R-warfarin. A noncompartmental approach was used for PK analysis using Phoenix WinNonlin (Version 6.3; Pharsight, Mountain View, CA, USA). The PK parameters determined for lobeglitazone included area under the plasma concentration– time curve in one dosing interval at steady state (AUCτ,ss), maximum observed plasma concentration at steady state (Cmax,ss), time to reach maximum plasma concentration at steady state (Tmax,ss), and t1/2. The PK parameters determined for R- and S-warfarin included area under the curve from time zero to the time of the last quantifiable concentration (AUClast), area under the curve from time zero to infinity (AUCinf), maximum observed plasma concentration (Cmax), Tmax, and t1/2. BODY.PD ASSESSMENT: Warfarin produces an anticoagulant effect by inhibiting hepatic vitamin K epoxide reductase, which is important for the activation of various coagulation factors including II, VII, IX, and X. The INR is the most widely used measure to determine the clotting tendency of blood after warfarin administration. Hence, the PD of warfarin was assessed by measuring the INR values and factor VII activity. The PD parameters for INR and factor VII activity determined included area under the effect–time curve from time 0 to 168 hours (AUEC0–168 h), baseline-corrected AUEC0–168 h, maximum effect (Emax) and time to reach maximum effect (TEmax). BODY.SAFETY ASSESSMENT: Safety was assessed by physical examinations, vital signs, clinical laboratory evaluation (hematology, coagulation, blood chemistry, and urinalysis), ECG, and by monitoring of adverse events (AEs). AEs were monitored throughout the study and were evaluated in terms of seriousness (serious, not serious), duration, intensity (mild, moderate, severe), outcome, and relationship with the study drug. All subjects who took at least one dose of the study drug were included in the safety analysis. BODY.STATISTICAL ANALYSIS: Statistical analyses were performed using SAS version 9.1.3 (SAS Institute, Cary, NC, USA). A mixed-effect model was applied to the log-transformed PK and PD parameters. Summary statistics and 90% confidence intervals (CIs) of the AUEC0–168 h and Emax for INR and factor VII geometric mean ratios (GMRs) (warfarin + lobeglitazone: warfarin alone) were provided for the evaluation of the PD interaction. If the 90% CIs of the Cmax and AUClast of the GMRs (warfarin + lobeglitazone vs warfarin or lobeglitazone alone) were within the range of 0.80–1.25, the absence of a PK interaction was concluded. BODY.RESULTS: BODY.STUDY POPULATION: Twenty-four subjects were randomized, and their mean (range) values for age and body mass index were 29.8 (range: 21–47) years and 22.5 (range: 19.1–26.6) kg m−2, respectively. Twenty-three subjects completed the study, and one subject withdrew for personal reasons during period 2. BODY.PK ASSESSMENT: Mean plasma concentration–time curves for R- and S-warfarin following administration of warfarin alone and when combined with lobeglitazone are shown in Figure 1. Cmax, Tmax, and t1/2 for R- and S-warfarin did not differ significantly between the two treatments (Table 1). The 90% CIs for the ratios of AUClast and Cmax of R-warfarin and S-warfarin for the coadministration of warfarin with lobeglitazone vs warfarin alone were within the predetermined no-interaction range (0.80–1.25). Steady-state plasma concentrations of lobeglitazone were achieved by day 4. None of the PK parameters for lobeglitazone were affected by the coadministration of lobeglitazone and warfarin (Figure 2). The GMRs and 90% CIs for the ratios of AUCτ,ss and Cmax,ss of lobeglitazone for coadministration of warfarin with lobeglitazone vs lobeglitazone alone were 0.9728 (90% CI: 0.9248, 1.0233) and 1.0247 (90% CI: 0.9671, 1.0856), respectively. BODY.PD ASSESSMENT: Baseline INR and factor VII levels were within ±5% of the normal range. Mean effect–time profiles over time when warfarin (25 mg) was administered alone and coadministered with lobeglitazone (0.5 mg) are shown in Figure 3. Summary statistics for the INR and factor VII activity following the administration of warfarin with and without lobeglitazone are shown in Table 2. Median INR levels increased to a maximum at 23.93 hours and 24.00 hours after administration of warfarin with and without lobeglitazone. About 168 hours after dosing, these values returned to the baseline under both treatments. Administration of warfarin with lobeglitazone did not affect the AUEC0–168 h or Emax of INR. The GMRs and 90% CIs for AUEC0–168 h and Emax for the INR were 1.0091 (90% CI: 0.9872, 1.0314) and 1.0003 (90% CI: 0.9675, 1.0342), respectively. Mean (standard deviation [SD]) baseline factor VII activities were 100.91% (SD: 15.68) for warfarin + lobeglitazone and 108.33% (SD: 16.23) for warfarin alone (P=0.0198; Wilcoxon rank sum test). Factor VII activity decreased to 17.70% at 23.90 hours and to 15.92% at 24.00 hours following administration of warfarin with and without lobeglitazone. Thereafter, factor VII activity reached the baseline level by 144 hours after dosing under both treatments. The observed GMRs and 90% CIs of AUEC0–168 h and Emax for factor VII were 0.9355 (90% CI: 0.9028, 0.9695) and 1.0935 (90% CI: 0.9934, 1.2037), respectively. This indicates that lobeglitazone had no effect on the PD of warfarin. BODY.SAFETY: Lobeglitazone was generally well tolerated when coadministered with warfarin in these healthy subjects. No serious AEs or AEs leading to discontinuation occurred in this study. Thirteen subjects (54%) reported a total of 28 treatment-emergent AEs, all of which were mild or moderate in severity. Among these AEs, three events reported by two subjects were treatment related: headache in the lobeglitazone-alone group, and epistaxis and nausea in the coadministration group. Epistaxis occurred 2 days after dosing in one subject whose INR value was <2.0 and who recovered without any medication. No clinically significant findings for vital signs or physical examination results were observed. BODY.DISCUSSION: The prevalence of diabetes is increasing, and aging of the population is the main driving factor for this increase in prevalence.13 Because older people with diabetes tend to have multiple comorbidities in addition to the traditional cardiovascular complications, it is likely that concomitant medications including warfarin may be used in these populations. Many studies have shown drug–drug interactions between warfarin and a wide variety of medications, including anticoagulants, antibiotics, and antidepressants, which are metabolized by hepatic microsomal enzymes. Salicylates potentiated the anticoagulant effect of warfarin, possibly because of their warfarin-like activity.14 Antibiotics such as trimethoprim and sulfamethoxazole potentiated the effect of warfarin by the inhibition of S-warfarin clearance.15 Selective serotonin reuptake inhibitor antidepressants, such as fluoxetine and fluvoxamine, impaired platelet aggregation by the inhibition of cytochrome P450 (CYP) 2C9-, CYP2C19-, and CYP1A2-mediated warfarin metabolism.16 Some TZDs also have shown a drug–drug interaction when used with warfarin. Troglitazone increased the INR in patients on concurrent warfarin therapy, which was theorized to be due to displacement of warfarin from plasma proteins or inhibition of the CYP system by troglitazone.12 Pioglitazone, a weak inducer of CYP3A4, significantly decreased patients’ INR.11 A case of INR increase was also reported on concurrent rosiglitazone therapy, which has no inhibitory effect on CYP2C9, 2C19, and 1A2 substrates.11 Due to the possibility of lobeglitazone being used concomitantly with warfarin in patients with type 2 diabetes mellitus, this open-label, crossover study to investigate potential clinical drug–drug interactions between lobeglitazone and warfarin was conducted. Lobeglitazone was administered at the approved daily dose of 0.5 mg for 4 consecutive days to reach steady state. Warfarin was administered during the steady state of lobeglitazone and, to maximize the interaction potential, the dosing of lobeglitazone continued until the effect of warfarin was maintained. In warfarin–drug interaction studies, warfarin is usually administered as a single large dose (eg, 25 mg) because the higher single dose of warfarin provides more opportunities to detect an interaction and reduces the exposure of healthy volunteers to a prolonged period of anticoagulation.17 In vitro assessment indicates that lobeglitazone is metabolized mainly by CYP3A4 and that its major metabolite, M7 (O-demethylation), is metabolized by CYP3A4, CYP2C19, and CYP2D6. Warfarin is eliminated almost entirely by metabolism; S-warfarin is metabolized to S-7-hydroxywarfarin mainly by CYP2C9, whereas R-warfarin is primarily metabolized by CYP1A2 and CYP3A4, yielding 6-, 8- and 10-hydroxywarfarin.18 S-warfarin is about twice as active as R-warfarin but is eliminated more rapidly.19 The exposure to lobeglitazone in this study is similar to that in a published study using the same dose.4 The observed PK profiles of R- and S-warfarin in the absence of lobeglitazone are also consistent with those reported in previous studies.20,21 In this study, the 90% CIs for the GMRs (warfarin + lobeglitazone vs warfarin) of Cmax and AUClast for R- and S-warfarin were within the bioequivalence guidelines-specified comparability bounds of 0.80–1.25. The 90% CIs were also entirely within the tightened interval of 90.00%–111.11% for narrow therapeutic index drugs.22,23 In line with the lack of an effect on warfarin PK, concomitant administration of lobeglitazone had no effect on the PD of single-dose warfarin, which was assessed through the measurement of INR and factor VII. The 90% CIs for the GMRs (warfarin + lobeglitazone vs warfarin) of INR and factor VII were within the range of 0.80–1.25. Because of the observed baseline differences in factor VII, baseline correction was also performed in the analysis. The 90% CIs for the GMRs of baseline-corrected AUEC0–168 h for factor VII were 0.9065–1.1088 (Table 2). As a result, coadministration of lobeglitazone is unlikely to be associated with an increase in warfarin-associated bleeding risk. The results of this study imply that lobeglitazone does not inhibit CYP2C9 and CYP3A4, the main enzymes involved in the metabolism of R- and S-warfarin. These findings may be applicable to other drugs metabolized by these pathways, suggesting that the coadministration of lobeglitazone with other drugs that are CYP2C9 or CYP3A4 substrates is unlikely to cause a clinically significant PK drug interaction. This study has a limitation. All participants were healthy young males, which is not typical of cardiovascular patients seen in the clinical setting. There was no sex difference for systemic lobeglitazone exposure at a 2 mg dose, which is four times the approved maximum dose of 0.5 mg.24 Because the anticoagulant response to warfarin is affected by several factors, the warfarin response must be monitored carefully even in the absence of drug interactions. BODY.CONCLUSION: In summary, concomitant administration of lobeglitazone and warfarin was generally well tolerated in healthy subjects. There were no significant changes in the PK or PD of warfarin when a single dose of warfarin was coadministered with repeated once-daily doses of lobeglitazone. Therefore, dose adjustment of warfarin is not required when these two drugs are administered in clinics.

TITLE: Anesthesiologist's satisfaction using between cisatracurium and rocuronium for the intubation in the anesthesia induced by remifentanil and propofol ABSTRACT.BACKGROUND: Although cisatracurium has many advantages in anesthetic practices, the best choice of a nondepolarizing neuromuscular blocking agent that can replace succinylcholine is rocuronium. However, it is reported that remifentanil with propofol might provide reliable intubating condition, even without a neuromuscular blocking agent; therefore, it might improve the intubating condition with cisatracurium. This study examined intubating conditions after administering rocuronium or cisatracurium in a rapid sequence induction with remifentanil-propofol. ABSTRACT.METHODS: Fifty two ASA physical status 1 or 2 adult patients scheduled for an elective surgery were enrolled in a randomized double-blinded trial. Anesthesia was induced in all patients with propofol 2.0 mg/kg and remifentanil 0.5 µg/kg, administered over 60 seconds. Rocuronium 0.9 mg/kg (3 × ED95, R group, n = 23) or cisatracurium 0.15 mg/kg (3 × ED95, C group, n = 29) was administered after the induction sequence. Laryngoscopy was attempted when the anesthesiologist thought it was 90 seconds after drug administration and appropriate time for intubation. The examiner, another anesthesiologist, recorded the exact time to intubation and suppression of maximal T1 on TOF. The intubating condition was assessed by the first anesthesiologist, as excellent, good, poor or not possible. ABSTRACT.RESULTS: The best time to laryngoscopy was predicted by measuring TOF and was found to be significantly longer in the C group (197 ± 53 s) than in the R group (102 ± 49 s) (P value < 0.05). However, time to larygoscopy, intubating condition during the laryngoscopy, and hemodynamic changes after intubation was similar in both groups. ABSTRACT.CONCLUSIONS: Despite fundamentally slower onset time, cisatracurium can provide quite good intubating conditions, which were comparable to those achieved with equipotent doses of rocuronium, which is more expensive in anesthesia inducted with remifentanil and propofol. BODY.INTRODUCTION: An ideal neuromuscular blocking agent needs to take the shortest time in endotracheal intubation, the best intubating condition and have the shortest duration of muscle paralysis [1]. Particularly, the rapid sequence induction of anesthesia is indicated in emergency situations in the presence of full stomach or other conditions with an increased risk of aspiration. In this point, rocuronium has the most rapid onset time among the currently available nondepolarizing neuromuscular blocking agents [2]. So rocuronium has become one of the most popular non-depolarizing neuromuscular blocking agents in clinical anesthesia. Rocuronium is a neuromuscular blocking agent with intermediate action of duration, a bolus dose of 0.6 mg/kg produces a clinically acceptable intubating condition in 60-90 s in most children, adults and elderly patients [3]. However, because rocuronium was mostly metabolized in the liver and excreted through bile [4], the duration of neuromuscular blockade of rocuronium may be prolonged in patients with liver and kidney failure [5,6]. Cisatracurium, the isomer of atracurium, is another kind of non-depolarizing neuromuscular blocking agent with intermediate action of duration. It is metabolized by Hoffmann elimination to laudanosine and a monoquaternary acrylate, like as atracurium [7]. Laudanosine has CNS stimulating properties. It is dependent on the liver and kidney for its elimination, and its concentration is elevated in patients with liver disease [8,9]. Cisatracurium is approximately three to four times as potent as atracurium. But cisatracurium produces laudanosine about five times less than atracurium does, and accumulation of this metabolite is not thought to be of any consequence in clinical practice [10]. In addition, cisatracurium does not release histamine in the range of clinical dose [11]. In spite of these advantages, the use of cisatracurium is limited because of slow onset and unsatisfactory intubating condition, compared with those seen with equipotent dose of other neuromuscular blocking agent [12-14]. Many studies have previously shown that endotracheal intubation may be accomplished without a neuromuscular blocking agent when anesthesia is induced with propofol and a short-acting opioid, such as remifentanil [15-19]. It is concluded that remifentanil and propofol might improve intubating condition, even if neuromuscular blocking agent is not used. As such, we hypothesized that pretreatments of remifentanil and propofol before the administration of cisatracurium might provide an acceptable intubating condition comparable to that of rocuronium. BODY.MATERIALS AND METHODS: After obtaining approval from the Institutional Review Board and informed written consent from patients (52 American society of anesthesiologists 1 or 2 patients) aged 27-61, undergoing general anesthesia and endotracheal intubation for elective surgery, were enrolled in the study. Exclusion criteria were the history of drug or alcohol abuse, gastroesophageal reflux or hiatus hernia, cardiovascular disease, reactive airway disease, body mass index 30 or more, allergies to drugs frequently used in anesthesia, administration of sedative or narcotic drugs in previous 24 hr, renal or hepatic impairment, or higher than class II of Mallampati classification. All patients were premedicated orally with 0.1 mg/kg of midazolam, 60 minutes before the induction of anesthesia. Before arriving at the operating room, patients had an IV catheter placed to allow administration of fluids and drugs. Patients were randomized using a random number generator to one of the two study groups to receive the following in a double-blinded manner. Standard monitoring, including noninvasive arterial pressure, ECG and pulse oximetry was applied and assessed continuously. Before induction of anesthesia, surface electrodes were placed over the ulnar nerve at the wrist. After preoxygenation with 100% oxygen, anesthesia was induced with 2.0 mg/kg of intravenous propofol, given over 30 s immediately followed over 30 s of 0.5 µg/kg of remifentanil IV injection. After the loss of consciousness, the ulnar nerve was stimulated at the wrist with a square wave stimulus set at a current of 50 mA and duration of 0.2 ms [20]. Each stimulus was delivered in a train-of-four (TOF) sequence, and repeated every 12 s using a TOF Guards® (Organon Teknika NV, Boxtel, Netherlands). The R group (n = 23) received 0.9 mg/kg of rocuronium (3 × ED95 of rocuronium), and the C group (n = 29) received 0.15 mg/kg of cisatracurium (3 × ED95 of cisatracurium), after the induction sequence. Rocuronium and cisatracurium syringes were prepared by an independent anesthesiologist in a total volume of 10 ml with normal saline. We performed endotracheal intubation when the anesthesiologist thought it was 90 seconds after the drug administration and appropriate time for intubation. The anesthesiologist was skilled and was not involved in the anesthesia technique, and he was blinded to the neuromuscular blocking agent's type. The examiner, another anesthesiologist, recorded the time to intubation and suppression of maximal T1 on TOF. To avoid vocal cord injury, endotracheal intubation was not attempted if the vocal cords were fully closed. After intubation, anesthesia was maintained with 50% nitrous oxide in oxygen, and 2.0 vol% sevoflurane (inspired). Intubating conditions were graded using the scoring scale initially described by Goldberg et al. [21] (Table 1). The onset time was defined as the time interval from the end of neuromuscular blocking agent administration to the maximal suppression of T1%. Measurements of the heart rate (HR) and the mean arterial pressure (MAP) were made immediately before induction, immediately before tracheal intubation, and 1 min after tracheal intubation. Statistical analysis was performed by using SPSS version 16.0 (SPSS Inc, Chicago, IL, USA). Quantitative data were expressed as the means ± SD, while qualitative data were expressed as numbers and percentages (%). Student t-test was used on parametric data (age, weight, mean blood pressure, heart rate, onset time), paired within groups and unpaired between groups. The Mann-Whitney U-test was used for the interval data (intubating scores) and the Chi-squared test for nominal data (sex). Probability value (P value) < 0.05 was considered statistically significant. BODY.RESULTS: There were no significant differences in patient demographics between the two groups (Table 2). The endotracheal intubation was successful on the first attempt in all patients. The overall intubating conditions did not differ between the two groups (P = 0.07). Intubating conditions were excellent in 22 patients in the R group and 24 patients in the C group (Fig. 1). Only one patient in the C group had poor intubating conditions. The remainder was rated as good. The various intubating scores are shown in Fig. 1, with no statistical significance between the two groups. The estimated time to intubation was similar in both groups (118.0 ± 34.6 and 111.8 ± 23.7 seconds in the R and the C groups, respectively). But the real onset time was significantly longer in the C group (197 ± 53 s), compared with that of the R group (102 ± 49 s) (P < 0.05; Table 3). Hemodynamic changes within each group are summarized in Table 4. The baseline values for HR were similar in both groups (71 ± 13 and 68 ± 14 mmHg in the R and the C groups, respectively). The HR recorded values of before intubation were 66 ± 12 and 62 ± 11 mmHg, respectively, with no statistically significant difference between the groups. After intubation of values for HR recorded were 85 ± 17 and 77 ± 16 mmHg, respectively, with no statistically significant difference between the groups. And the HR decreased from the baseline in response to administration of induction agents in both groups, and increased from the baseline in response to intubation in both groups. The baseline values for MBP were similar in both groups (94 ± 9 and 90 ± 10 mmHg in the R and the C groups, respectively). The MBP values before intubation were 83 ± 11 and 75 ± 11 mmHg, respectively, with no statistically significant difference between the groups. The MBP values after intubation were 103 ± 23 and 95 ± 16 mmHg, respectively, with no statistically significant difference between the groups. And the MBP decreased from the baseline in response to administration of induction agents in both groups, and increased from the baseline in response to intubation in the R groups, but showed no significant change in group C. BODY.DISCUSSION: Cisatracurium has many advantages, compared with other neuromuscular blocking agent. Cisatracurium is a nondepolarising neuromuscular blocking agent with an intermediate duration of action. It is the cis isomer of atracurium besilate, and is approximately 3 to 4-fold more potent than the mixture of isomers that constitute the parent drug. However, cisatracurium produces laudanosine about five times less than atracurium, and accumulation of this metabolite is not thought to be of any consequence in clinical practice. Relative to atracurium, cisatracurium has a lower propensity to cause histamine release, which is more potent but has a slightly longer onset time at equipotent doses [7]. And compared with vecuronium, cisatracurium besilate was associated with a significantly faster recovery after continuous infusion in patients in intensive care. It also offers a more predictable recovery profile than vecuronium after prolonged use in patients in intensive care [8,9]. In our results, the onset time was determined as the time interval from the end of neuromuscular blocking agent administration to the maximal suppression of T1%. In patients undergoing elective surgery, our report showed that the onset time of cisatracurium (197 ± 53 s) was significantly longer compared to rocuronium (102 ± 49 s). Nonetheless, we performed endotracheal intubation when the anesthesiologist thought it was 90 seconds after the drug administration and appropriate time for intubation. The estimated time to intubation was delayed, similar in both groups, from 90 seconds (118.0 ± 34.6 and 111.8 ± 23.7 seconds in the R and the C groups, respectively). We can get excellent or good tracheal intubating conditions in 96% of patients from a dose of cisatracurium 0.15 mg/kg, which is similar to those achieved by 0.9 mg/kg of rocuronium (100%), while supplementing a remifentanil and propofol combination. We thought most of anesthesiologists can adjust the intubation time from the drug administration with the sense of mask ventilation. The estimated intubation time from our result seems to be a nonscientific or just an empirical method in anesthetic practice, but there is no case who cannot attempt if the vocal cords were fully closed. So, we can conclude the maximal suppression of T1 in TOF is not a good indicator for intubation from our results. Although cisatracurium is more potent than the parent mixture (95% effective dose [ED95] 0.05 mg/kg vs. 0.2 mg/kg), its pharmacodynamic profile is similar to that of atracurium, except for a reportedly slower onset [6]. Bluestein et al. [22] reported that increasing the initial dose of cisatracurium (from 0.1 to 0.15 and 0.2 mg/kg), decreased the mean time of onset (from 4.6 to 3.4 and 2.8 min, respectively) and increased the mean time of clinically effective duration (45 to 55 and 61 min, respectively). Mellinghoff et al. [12] estimated that the onset time was 3.1 ± 1.0 min with cisatracurium. Mandal [23] conducted a study to find out about the minimum possible dose of cisatracurium for achieving excellent to good intubating conditions within 90 s of its administration under general anesthesia. After induction of anesthesia with the standardized method, group I (n = 20) received 0.15 mg/kg, group II (n = 20) received 0.2 mg/kg and group III (n = 20) received 0.25 mg/kg of cisatracurium. They concluded that the minimum dose required to achieve excellent to good intubating conditions with cisatracurium is 0.20 mg/kg at 90 s after its administration. Doses of 0.15 mg/kg (3 × ED95) and 0.2 mg/kg (4 × ED95) of cisatracurium, as components of a propofol/nitrous oxide/oxygen induction intubation technique, may produce generally good or excellent conditions of intubation in 2.0 and 1.5 min, respectively [24]. In accordance with these results, we can conclude that at the same dose (3 × ED95) atracurium would not create a superior onset time and intubating condition, compared with rocuronium after 90 s. On the other hand, recent studies have suggested that propofol in combination with short-acting opioid, such as alfentanil or remifentanil, may provide adequate conditions for laryngoscopy and endotracheal intubation, without using neuromuscular blocking agents for several properties of these drugs. First of all, remifentanil has a property of analgesic efficacy and respiratory depressant effect. The opioid remifentanil is a potent fentanyl derivative. Remifentanil has unique properties and undergoes rapid hydrolysis. It has a rapid termination of actions due to hydrolysis by non-specific tissue esterases with a context sensitive half-life of just over 3 min, allowing rapid return of spontaneous respiration. Although the onset of effect is similar to that of alfentanil, within one to two minutes, it has a shorter half-life and the time to recovery is not greatly influenced by the dose [22]. Those clinical properties make remifentanil the short-acting opioid of choice for circumstances, in which an intense opioid effect of short duration is required. Based on its analgesic efficacy and its respiratory depressant effect, remifentanil is 20 to 30 times more potent than alfentanil, after a single bolus dose [22]. In a recent study, Stevens and Wheatley [18] showed that when used in combination of 2 mg/kg of propofol, 3-4 µg/kg of remifentanil provides satisfactory intubating conditions, more reliably than 1-2 µg/kg of remifentanil. But a high dose of opioid would cause a prolonged apnea and a fatal decrease in arterial blood pressure, during induction of anesthesia. So in our study, the low dose was chosen in an attempt to minimize the effects on apnea time and cardiovascular parameters. In the second place, propofol has a property of depressant effect on pharyngeal and laryngeal muscles. The vocal cord sequelae occur more frequently in patients for whom tracheal intubation is attempted in a situation of unacceptable neuromuscular blocking. Supplementing a propofol-opioid induction regimen with a non-depolarizing neuromuscular blocking agent improves the quality of endotracheal intubation and decreases postoperative hoarseness [24]. McKeating et al. [25] investigated the depressant effects of induction doses of thiopentone and propofol on airway integrity and reactivity. They found that, when no neuromuscular blocking agents were given, laryngoscopy was easier to perform after propofol than after an equipotent dose of thiopentone, and that pharyngeal and laryngeal activity were more depressed after administration of propofol than after of thiopentone. Using a fiberoptic laryngoscope, Barker et al. [26] assessed the vocal cord movement after induction of anesthesia with either propofol or thiopentone, and observed that vocal cords adducted to a greater extent after induction of anesthesia with thiopentone than with propofol. More recently, Eames et al. [27] compared the effects of etomidate, propofol and thiopentone on respiratory resistance after tracheal intubation. Anesthesia was induced with either 2.5 mg/kg of propofol, 5 mg/kg of thiopentone or 0.4 mg/kg of etomidate. Respiratory resistance after tracheal intubation was lower after induction with propofol than after induction with thiopental or with high-dose etomidate. In accordance with these results, we found that pharyngeal and laryngeal activities were more depressed after the administration of propofol than after thiopentone or etomidate, although, we used a relatively low dose of propofol. These findings should be considered within the context of the limitation of this study. First, the non-treated control group, without remifentanil, was not included in this study. However, because we performed endotracheal intubation when the anesthesiologist thought it was 90 seconds after the drug administration and appropriate time for intubation at earlier point compared with usual onset time of cisatracurium, including a non-treated arm would not have been ethical. Second, we did not use the higher doses of remifentanil. Higher doses of remifentanil would possibly produce further improvement in intubating condition. This will be of clinical benefit if it is not associated with an increase in the incidence of complications. In conclusion, our report shows that the onset time of cisatracurium was significantly longer, compared to rocuronium. However, a small dose of 0.15 mg/kg of cisatracurium, supplementing a remifentanil-propofol combination, provides excellent or good endotracheal intubating conditions in 96% of patients, a rate similar to those achieved by rocuronium 0.9 mg/kg (100%), while using the same anesthetic technique and similar induction time. With such technique, we were able to reproduce the advantages of cisatracurium, such as non-accumulation of metabolite. This technique could be applied in patients with liver and kidney failures for brief surgical procedures to obtain short time to intubation and excellent intubating conditions.

TITLE: Coffee intake and development of pain during computer work ABSTRACT.BACKGROUND: The present study sought to determine if subjects who had consumed coffee before performing a simulated computer office-work task found to provoke pain in the neck and shoulders and forearms and wrists exhibited different time course in the pain development than the subjects who had abstained from coffee intake. ABSTRACT.FINDINGS: Forty eight subjects all working fulltime, 22 with chronic shoulder and neck pain and 26 healthy pain-free subjects, were recruited to perform a computer-based office-work task for 90 min. Nineteen (40%) of the subjects had consumed coffee (1/2 -1 cup) on average 1 h 18 min before start. Pain intensity in the shoulders and neck and forearms and wrists was rated on a visual analogue scale every 15 min throughout the work task. During the work task the coffee consumers exhibited significantly lower pain increase than those who abstained from coffee. ABSTRACT.CONCLUSIONS: Subjects who had consumed coffee before starting a pain provoking office work task exhibited attenuated pain development compared with the subjects who had abstained from coffee intake. These results might have potentially interesting implications of a pain-modulating effect of caffeine in an everyday setting. However, studies with a double blind placebo controlled randomized design are needed. BODY.FINDINGS: BODY.BACKGROUND: Shoulder and neck pain occur commonly during work involving very low levels of muscle activity, such as office work with computers, with prevalence rates around 10 % [1-3]. Recently, we have reported that computer office-work performed continuously for 90 min, with time pressure and high precision demands, induced substantial pain in the shoulders and neck as well as in the forearm operating the computer mouse both in subjects with chronic pain and in healthy references [4,5]. Of the participants in this study, nearly half of them had ingested coffee before attaining the laboratory experiment. Reduced muscle pain after caffeine administration have been reported during dynamic exercise of >60% of maximal capacity [6,7] and during static grip to exhaustion tasks [8]. The present study sought to determine if subjects who had consumed coffee before starting the computer office-work exhibited different time course in the pain development than subjects who had abstained from coffee intake. BODY.MATERIALS AND METHODS: BODY.SUBJECTS: Forty-eight subjects, 22 with chronic shoulder and neck pain (pain group) and 26 healthy pain-free subjects (reference group), all working fulltime, were recruited through advertisements in local papers and the Internet (for characteristics, see [4,5]). All participants received written information and signed an informed consent. The Norwegian Regional Committee for Medical Research Ethics and the Norwegian Social Science Data Services approved the study. BODY.EXPERIMENTAL PROTOCOL: The present study is part of a larger study investigating mechanisms of pain development during office work, however not the effect of caffeine. Extended details of the experimental protocol and procedures are presented elsewhere [4,5]. In short, the subjects had to report to the laboratory between 8 and 9 a.m. at the experimental day. To avoid decreased vigor and alertness, sleepiness, and fatigue, as have been reported when missing the regular morning coffee, the participants had the opportunity to ingest coffee or tea in conjunction with a light breakfast before attaining the laboratory. If they chose to consume coffee, they were instructed to drink no more than one cup. They were also instructed to avoid taking any kind of medication, using alcohol, and performing exhaustive physical activity the 24 hours before the experiment. When reporting to the laboratory it was registered if they had consumed coffee (yes/no), how much, and at what time. It was however not registered if they were a habitual coffee user or not. The experimental protocol consisted of a computer-based office-work task including correcting typographical errors in a standardized text using a word processor as fast and accurately as possible for 90 min without pauses followed by 30 min recovery [4,5]. The participants were allowed to use the computer mouse only. Measurements included ratings of current pain intensity in the right and left shoulder and neck and forearms and wrists on a visual analogue scale (VAS). The VAS comprised a 100 mm line anchored by the labels “no pain”; 0, and “unbearable pain”; 100 (in Norwegian “uutholdelig smerte”, a phrase commonly used in the Scandinavian language). After baseline measurements VAS-ratings were rated every 15 min throughout the work task and recovery. For extended details on the procedures and measurements, see Strøm et al. [4,5]. BODY.STATISTICS: Statistical analyses were performed using the Statistical Package for the Social Sciences (release 18.0, SPSS Inc., Chicago, IL, USA). Independent sample t test was used to explore between-group differences in anthropometrics. A two-tailed significance level of 5% was adopted. To fit the best model for the time course of the dependent variable pain for the coffee consumers and coffee abstainers linear mixed model analysis was used [9]. The time was treated in the model as a continuous factor. The models were fitted either with an unstructured or a heterogeneous first-order autoregressive covariance structure based on Akaike’s information criterion for goodness of fit [9]. Separate analyses were performed to check for the influence of group (pain and reference groups). BODY.RESULTS: Nineteen (40 %) of the 48 subjects reported that they had consumed coffee before attaining the laboratory (8 subjects in the pain group and 11 subjects in the reference group). The volume of coffee ingested ranged from ½ a cup to 1 cup (median 1). The mean time from coffee consumption until the experimental start was 1 h 18 min ± 28 min. No statistically significant differences were found between the coffee consumers and the coffee abstainers regarding age, bodyweight and –height. Both the coffee consumers and abstainers exhibited pain in the shoulders and neck and forearms/wrists during the work task (Figure 1). The pain increase in the active side of the shoulder/neck was significantly lower for the coffee consumers than for the abstainers (see Table 1; β5), and at the work task end the mean pain intensity was 41 ± 22 mm and 55 ± 29 mm for the consumers and non-consumer, respectively. Similar results for the pain increase were found for the inactive shoulder and neck, and the forearms/wrists. The differences in time course between the coffee consumers and abstainers were still statistically significant (p<0.05), when including the presence of chronic pain or not (i.e. pain and reference groups). Figure 1Pain intensity. Mean values (± SEM) of pain intensity in (a) the active side of the shoulder and neck, (b) the inactive shoulder and neck, (c) the active forearm and wrist, and (d) the inactive forearm and wrist, rated on a visual analogue scale (VAS, mm) at baseline (0) and every 15 min throughout the 90 min office work task and 30 min recovery, in subjects that had consumed coffee before start (n = 19; open circles), and in subjects that had abstained from coffee consumption (n = 29; filled circles). The shaded area indicates the 90 min work task period. Table 1Time course of pain intensity  Estimate of the βb95% CIp-value(β1) Intercept 7.5 5.2 9.8 < 0.001 (β2) Coffeeic −3.5 −7.2 0.2 0.064 (β3) Timeijd 7.3 5.9 8.7 < 0.001 (β4) BP90ij −16.6 −21.1 −12.1 < 0.001 (β5) Coffeei * Timeij −2.9 −5.1 −0.7 0.011 (β6) Coffeei * BP90ij 3.4 −3.8 10.6 0.34 Estimated regression coefficients with 95% confidence intervals (CI) based on the fitted linear modelsa of the pain intensity ratings (VAS, mm) in the active side of the shoulder and neck during and after performing the 90 min computer work in subjects that had consumed (n = 19) or abstained (n = 29) from coffee before start. aLinear mixed models for repeated measurements fitted with unstructured covariance structure. bβ2: the difference between coffee consumers and abstainers at baseline; β3: the change per 15 min time period during the 90 min work task; β4: BP90 = break point 90 min, i.e. the change from the work task ending and through each 15 min period during the 30 min recovery; β5: the difference between coffee consumers and abstainers for each 15 min period during the work task; β6: the difference between coffee consumers and abstainers per 15 min periods during the recovery. c0 = No; 1 = Yes. d0 = Baseline; 1 = 15 min; 2 = 30 min, and so on. BODY.DISCUSSION: The present results revealed significant lower increase in the pain development during the computer office-work task for the subjects who had consumed coffee approximately 1 ½ h before the task than subjects that had abstained from coffee. The present study was originally undertaken to investigate mechanisms of pain development during computer office work [4,5], and not the effect of coffee on pain. Thus, the present results must be viewed within the limitations of the study regarding the effect of the coffee consumption. Since a controlled randomized design of the coffee consumption was not used a lot of uncertainties about the association between coffee intake and differences in pain-perception during the office work task exist. The exact dose of coffee is unknown; the quantity of coffee consumed was self-reported even though one cup was the maximal allowed limit; the size of a cup may vary and also the caffeine dose [10], and blood samples were not taken to measure the caffeine in the system. The time from coffee intake to experimental start was not standardized. The mean time spent from coffee consumption until start was, however, within the half-life of caffeine [10]. Nevertheless, the attenuated pain response seen for the coffee drinkers was statistically significant for all measured pain sites even when adjusted for the presence of chronic pain, and a difference in VAS pain score >14 mm can be considered as clinically significant [11]. Furthermore, the size of the effect of caffeine on pain attenuation in the active shoulder/neck indicates a moderate effect (Cohen’s d = 0.54). Thus, we hypothesize that the coffee consumption may have had an attenuating effect on the pain development during the computer work. The reason for allowing the coffee intake before starting the experiment was to avoid unpleasant effects of caffeine deprivation, e.g. decreased vigor and alertness, sleepiness, and fatigue, as have been reported when missing the regular morning coffee in habitual coffee drinkers [12,13]. It is reasonable to believe that those who ingested coffee before start were habitual coffee drinkers. However, it is not known if those who not ingested coffee also were habitual coffee users or not. We are not aware of studies that have examined the effect of coffee consumption on naturally occurring pain during work of very low-level muscle activity, as during computer work. However, several studies have reported attenuated muscle pain after caffeine administration during dynamic exercise of >60% of maximal capacity [6,7], and during ischemic [14] and eccentric muscle contractions [15]. The caffeine doses in these studies were >5 mg/kg, and probably higher than what is presumed doses in the present study. A single cup of coffee provides a caffeine dose of 0.4 to 2.5 mg/kg [10] and most of the caffeine is absorbed from the gastrointestinal tract within 45 min. For doses lower than 10 mg/kg the half-life of caffeine is between 2.5 and 4.5 h [10]. Caffeine is metabolized in the liver to form the methylxanthines paraxanthine and theophylline [16]. Caffeine is an adenosine receptor antagonist [10]. Adenosine receptors are distributed throughout the nervous system, in the vascular endothelium, heart, liver, adipose tissue, and in the skeletal muscle [17-19]. The potential mechanism of caffeine on the pain response in the presents study is uncertain. Caffeine has high affinity to both A1 and A2A receptors [10]. Adenosine may exert its influence on nociception via peripheral C-fiber involvement and within the central nervous system [20]. Activation of adenosine A1 receptors inhibits nociception both in the periphery and at the spinal level, while activation of A2A receptors augments nociception peripherally, but seems to have no direct effect on nociception at the spinal level [20]. The action of caffeine thus depends on the type of receptors that is blocked and on tissue in which the receptors are located. In conclusion, subjects who had consumed one cup of coffee before starting a pain provoking office work task exhibited attenuated pain development compared with the subjects who had abstained from coffee intake. These results might have potentially interesting implications of a pain-modulating effect of caffeine in an everyday setting. However, studies with a double blind placebo controlled randomized design are needed. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: All authors participated in the design and conception of the study. VS and CR conducted the laboratory work. VS analyzed the data and drafted the manuscript. All authors read, revised and approved the final manuscript.

TITLE: Influence of methylphenidate treatment assumptions on cognitive function in healthy young adults in a double-blind, placebo-controlled trial ABSTRACT.BACKGROUND: Increasing numbers of students use stimulants such as methylphenidate (MPH) to improve their study capacity, making them prone to subsequent prolonged drug abuse. This study explored the cognitive effects of MPH in students who either assumed they received MPH or assumed they received a placebo. ABSTRACT.METHODS: In a double-blind, randomized, placebo-controlled trial with a between-subjects design, 21 students were subjected to partial sleep deprivation, receiving no more than 4 hours sleep the night before they were tested. In the morning, they were given either a placebo or 20 mg of MPH. They then performed free recall verbal tests and Go/No-Go tasks repeatedly, their moods were evaluated using Profile of Mood States and their tiredness was assessed using a visual analog scale, with evaluation of vigilance. ABSTRACT.RESULTS: No significant differences were found between those subjects who received MPH and those who received a placebo. However, significant differences were found between subjects who assumed they had received MPH or had no opinion, and those who assumed they had received a placebo. At three minutes, one hour, and one day after memorizing ten lists of 20 words, those who assumed they had received MPH recalled 54%, 58%, and 54% of the words, respectively, whereas those who assumed they had received placebo only recalled 35%, 37%, and 34%. ABSTRACT.CONCLUSION: Healthy, partially sleep-deprived young students who assume they have received 20 mg of MPH experience a substantial placebo effect that improves consolidation of information into long-term memory. This is independent of any pharmacologic effects of MPH, which had no significant effects on verbal memory in this study. This information may be used to dissuade students from taking stimulants such as MPH during examination periods, thus avoiding subsequent abuse and addiction. BODY.INTRODUCTION: BODY.METHYLPHENIDATE: Methylphenidate (MPH) is an amphetamine analog that increases extracellular levels of noradrenaline and dopamine in the brain by blocking their monoamine transporters.1 An increase in extracellular dopamine is thought to improve attention and motivation, and to cause a general increase in motor activity.1 Noradrenaline is believed to increase wakefulness, alertness, exploratory activity, and attention, and also has an effect on mood and blood pressure.1,2 In spite of the emergence of newer drugs, MPH is still one of the most frequently used drugs in the treatment of attention deficit hyperactivity disorder (ADHD) in children and adolescents, and is recommended as first-line treatment for ADHD.3 During the last few years, there has been a significant increase in the diagnosis of ADHD and in the corresponding prescription of stimulant drugs such as MPH.1,4 Reported side effects of MPH are anorexia, nausea, vomiting, stomach ache, nervousness, headache, insomnia, and tachycardia; these effects increase linearly with the dose. Other adverse effects are motor and vocal tics.5,6 BODY.MEMORY IMPROVEMENT AFTER AMPHETAMINE USE: Several placebo-controlled studies have used free recall of word lists to examine the cognitive effects of d-amphetamine on healthy adults. These studies show that d-amphetamine does not influence short-term memory,7–10 but does act after initial memory encoding to improve long-term verbal memory. This improvement is prolonged by administering the drug before or during consolidation, as learned information is gradually stored into long-term memory.8 BODY.MISUSE OF STIMULANTS BY COLLEGE STUDENTS: Recent research shows that MPH misuse is increasing among college students, as is the illicit use of other stimulants normally used to treat ADHD; some studies report a 10-fold increase in the past two decades.6,11 University students also misuse stimulants: for example, 4% of US university students misuse MPH each year.12 The motivations that university students cite most frequently for stimulant use are to heighten concentration (58%), increase alertness (43%), and just to “get high” (43%).13 BODY.USE OF STIMULANTS DURING EXAMINATION PERIODS: Many students experience high stress levels and sleep deprivation during examination periods, which may impair their cognitive abilities. Each student handles this stress differently; sports, relaxation, vitamin supplements, tea, and coffee are all popular choices. However, a probably underestimated number of students rely on stimulant drugs such as MPH to study better, making them more prone to subsequent prolonged drug abuse.14 It is possible that MPH may have a substantial placebo effect on students; the mere thought that they have taken something to help them study may improve their cognition. It is interesting to note that, in a number of double-blind, placebo-controlled trials in various other domains, participants who assumed they had received an active treatment showed significant better outcomes than those who assumed they had received a placebo.15–20 BODY.AIM OF STUDY: There are numerous studies comparing stimulants with placebo, but far fewer in study groups without ADHD. Thereupon, only a limited number of studies have investigated the placebo effects of stimulants, and only a few of these have focused on MPH. These studies did show that the actual effectiveness of MPH did not live up to subjects’ prior expectations.21 An improvement of memory was found, but no consistent evidence for other enhancing effects was uncovered.21 However, the placebo effect with MPH is not well characterized, despite significant and increasing MPH use and misuse. Therefore, more studies are needed to clarify this effect. To our knowledge, no studies have examined the effect of a subject’s MPH “treatment assumption”, ie, whether they assume they have received MPH or a placebo, regardless of which they have actually received. The aim of this double-blind, placebo-controlled study was to investigate this treatment assumption effect by comparing the cognitive performance of healthy young students who believed they had received MPH with the performance of those who thought they had received a placebo or had no opinion. Subjects were partially sleep-deprived during the study to simulate examination period conditions. BODY.MATERIALS AND METHODS: BODY.SUBJECTS: Healthy young student volunteers were recruited using posters and advertisements in the university newspaper. Participants were excluded if they suffered from diabetes mellitus, hypertension, arrhythmias, thyrotoxicosis, epilepsy, ADHD, or any other disorder that could affect their ability to concentrate, such as sleep disorders or intellectual disabilities. Other exclusion criteria were as follows: pregnancy, a history of drug abuse, prior use of psychoactive substances including MPH, a history of depression, a family history of heart problems before the age of 60 years, and familial hyperlipidemia. In total, 21 subjects were recruited, including seven males and 14 females, of mean age 23 ± 3 (range 18–33) years. All these volunteers signed an informed consent document prior to participation in the trial. They were also asked to refrain from drinking any caffeinated or alcoholic beverages and to eat no more than two pieces of fruit in the 24 hours prior to drug administration, in order to avoid any influence on their metabolism of MPH. BODY.STUDY DRUG: The drug dose used was 20 mg of MPH in an immediate-release formulation. While this is twice the dose recommended for most therapeutic purposes, it is the most common dose used in earlier studies of the ability of MPH to enhance cognitive function in normal young adults.21–23 Identical, unmarked capsules were used for both the drug and the placebo; these were administered orally approximately one hour before the initial study phase of free recall testing. When administered orally, MPH is absorbed almost completely, and food has little impact on this process. In both adults and children, MPH reaches a peak concentration 1.5–2.5 hours after ingestion of a single, immediate-release dose, and has an elimination half-life of 2–3.5 hours.24,25 BODY.PROCEDURE: A randomized, placebo-controlled design was used, and was blinded for participants, dispensers, and outcome assessors. All subjects knew prior to enrollment that they were participating in a double-blind study that used MPH. The cognitive tests used in the trial were a free recall of words, a Go/No-Go task, and a vigilance assessment. Various tests were scheduled during and immediately after the initial free recall study phase, after a one-hour delay, and after a one-day delay. The evening before they were tested, the participants reported to the laboratory at 8:30 pm. On arrival, they were asked to answer a questionnaire, in order to exclude anyone who had, in the previous 24 hours, consumed any caffeinated or alcoholic beverages, any psychoactive substances, or more than two pieces of fruit. They also completed a brief physical examination, which included measurement of their blood pressure and heart rate. Trial conditions were designed to mimic an examination period, and thus included partial sleep deprivation. Subjects were told to remain awake until 2 am, and were given several popular games to act as cognitive distracters, ie, two pinball machines, one air hockey table, six Nintendo Wii™ consoles, a poker set, a ping-pong set, Mastermind™, and Roll-It™. Participants were also told not to eat and to drink nothing but water. At 2 am, the subjects were told to go to sleep in prepared rooms. They were then awakened at 6 am, at which time they all consumed a similar breakfast of croissants, sandwiches, jam, milk, and water. Immediately after breakfast, their baseline blood pressure, heart rate, and body temperature were measured. Approximately one hour before the start of free recall testing, participants were given either 20 mg of MPH or a placebo by oral administration. To determine which they should receive, they were randomized by age and gender using a computer-based minimization procedure. Of the 21 participants, ten received a placebo and 11 were given MPH. Prior to starting the study phase of the free recall test, participants were informed that they were going to be shown a series of words, which they must memorize for an unspecified memory test that they would be given at a later time. They were then shown ten lists, each containing 20 unrelated words. After seeing each list, they were given 2 minutes to write down as many words as they could remember from the list, in a test of their immediate free recall. The study phase lasted for approximately 35 minutes. After an additional 3 minutes, the participants were given 15 minutes to recall all 200 words, in a final free recall test. One hour after the end of the study phase, this test was repeated in a one-hour delay free recall (1HDFR), and was repeated again after one day (1DDFR).7 After completing the final free recall and 1HDFR, the subjects were asked what they assumed they had received, ie, a placebo, MPH, or “I don’t know”. As described previously, this was their “treatment assumption”. The results of the final free recall and 1HDFR were not known before their treatment assumption was questioned. Only at the end of the first day, the individual results were communicated to the participants. The Go/No-Go task was performed twice, ie, once directly after the final free recall (approximately 2.5 hours after administration of MPH or a placebo) and the second time directly after the 1HDFR (approximately 3.5 hours after administration). In both cases, each participant’s blood pressure, heart rate, and body temperature were measured directly afterwards. The schematic overview of the experimental procedure is shown in Figure 1. BODY.WORD LISTS: For free recall testing, ten lists of 20 unrelated Dutch words were used. These were taken from a previous cognitive experiment with d-amphetamine.7 Each word had one syllable and consisted of three to five letters. The 200 words were matched in frequency, following norms developed by Uit den Boogaart.26 These words were presented in the center of a computer screen at a constant rate of 4 seconds per word. The order of the words was randomized, but the same presentation order was used for all participants.7 During testing, subjects incorrectly “recalled” words that were not actually included in any the ten-word lists. These words were counted in this study and referred to as intrusions. The short-term memory recency effect was investigated by analyzing how well subjects recalled the last five words in each word list. The long-term memory recency effect was investigated by doing the same with the first five words in each word list. BODY.GO/NO-GO TASK: To measure a participant’s capacity for sustained attention and response control, a target-detection Go/No-Go task was replicated from earlier studies.23,27,28 Each subject was required to watch a computer screen that displayed a random series of two-digit numbers; each number was shown for 200 milliseconds and was followed by a 700 millisecond interstimulus interval. A target (Go) occurred when any number appeared twice in a row. During this task, which lasted approximately 21 minutes, one quarter of the numbers were targets. When a target (Go) appeared, the subjects were instructed to press any key on the keyboard as quickly as possible. They had to suppress this response when there was no target (No-Go). The computer registered how quickly participants responded to each Go target, from the time the target appeared until the time a key was pressed, as well as the number of correct hits. Reaction times were recorded to the nearest millisecond. BODY.VIGILANCE ASSESSMENT: Vigilance is the ability to maintain attention and alertness over a prolonged period of time. To assess this, the reaction times from the last 3 minutes of both Go/No-Go task runs were examined. The computer recorded these reaction times in milliseconds.23,29 BODY.SUBJECTIVE STATE TESTS: The Profile of Mood States (POMS) was used to record the subjective state of each participant and a visual analog scale (VAS) was used to measure tiredness. POMS consists of 51 adjectives, which subjects must rate on a scale of 0 to 4. These adjectives can be subdivided into the following variables: tension, depression, anger, fatigue, and vigor.29 For the VAS, a 100 millimeter horizontal line was used, anchored by the descriptors “not tired” at the left end (0) and “exhausted” (100) at the right end. Subjects used this VAS to rate the question “How tired/fatigued do you feel right now?”.30–32 The response value was measured in millimeters from the left end of the scale. Participants completed the POMS and VAS tests before drug administration, and at approximately 2 hours, 4 hours, and one day later. BODY.PRETEST AND POST-TEST QUESTIONNAIRE: When the participants arrived on the evening before testing, they were asked what they had eaten and drunk during the last 24 hours. Their weight and height were also noted, along with any sports they played and the number of hours they exercised each week. One week after the experiment, the participants were asked via email about the methods they used to memorize and recall the words in the free recall test. BODY.ETHICAL APPROVAL: The ethics committee of the University Hospital of Brussels, Vrije Universiteit Brussel, approved the experimental design of this study. BODY.STATISTICAL ANALYSIS: To assess the influence of treatment assumption on cognitive function, the study population was divided into three main groups based on their treatment assumption: placebo (subjects who assumed they had received a placebo when asked); MPH (subjects who assumed they had received MPH); and DNK (subjects who had no opinion, ie, “did not know”). A one-way analysis of variance was used to compare the participants who assumed they had taken a placebo with those who assumed they had taken MPH or said that they did not know. A criterion alpha level of 0.05 was used throughout the analyses. BODY.RESULTS: BODY.GENERAL RESULTS: The protocol was completed by 21 volunteers. On arrival, none of them had a blood pressure greater than 140/90 mmHg or an irregular heart rate. The characteristics of the population are shown in Table 1. No significant effects on cognition were observed between the MPH and the placebo group when comparing the immediate and delayed free recalls, Go/No-Go tasks, or vigilance tasks after administration of placebo or 20 mg of MPH. Subjects from both study groups remembered 42% of the words during the final free recall (right after study phase) and 44% during the 1HDFR. After the final free recall, subjects from the MPH group had 221 correct hits for the Go/No-Go task compared with 231 correct hits in the placebo group (P = 0.057); the vigilance test also did not show any significant difference in reaction times between the groups. According to our study results, MPH does not improve consolidation into long-term memory in healthy young adults with short-term sleep deprivation. Because there was no significant difference in performance between those on MPH and those on placebo, the treatment groups were combined and the results were reanalyzed by treatment assumption. BODY.FREE RECALL: Table 2 shows the mean number of words that subjects recalled correctly out of 200, depending on their treatment assumptions at final free recall and again at 1HDFR. Subjects who reported “MPH” or “DNK” after final free recall remembered significantly more words at final free recall, 1HDFR, and 1DDFR than those who reported “placebo”. There were no significant differences between those who reported “MPH”, “DNK” or “placebo” after 1HDFR, although the numbers still showed the same trend. These results, especially for treatment assumption after final free recall, contrast with the results from the real MPH and placebo groups, which showed no significant differences in correct word recall. BODY.NUMBER OF INTRUSIONS: Analysis showed that treatment assumptions made no significant difference to the number of intrusions. Furthermore, there were no significant differences in intrusions between the real MPH and placebo groups. BODY.RECENCY AND PRIMACY EFFECTS: Subjects recalled the last five words significantly better in the immediate free recall than in the final free recall, 1HDFR, or 1DDFR, since the words were still in short-term memory (Table 3). All applicable P-values were significant. In general, subjects who reported a treatment assumption of MPH or DNK recalled the last five words better than those who assumed placebo. There was, however, no difference between the real MPH and placebo groups. The long-term memory primacy effect was studied by comparing how well subjects recalled the first five and last five words in each word list, as shown once again in Table 3. In the final free recall, 1HDFR, and 1DDFR, subjects recalled approximately twice as many of the first five words in comparison with the last five words. In most cases, subjects whose treatment assumption was MPH or DNK recalled the first five words better than those who assumed they had received a placebo. Again, there were no significant differences between the real MPH and placebo groups. Subjects reported their treatment assumptions after final free recall and again after 1HDFR, but similar recency and primacy results were found no matter which set of treatment assumptions was used. BODY.GO/NO-GO TASK: Subjects performed the target detection Go/No-Go task twice, at 2.5 and 3.5 hours after administration of the drug. It should be noted that the results from two subjects (a man and a woman) were excluded, due to an error in data recording. During this 21-minute task, the drug did not influence the outcome. The real MPH and placebo groups performed similarly, registering 221 and 231 hits, respectively, in the Go/No-Go task that was performed after 2.5 hours; the corresponding reaction times were 347 and 345 milliseconds. Treatment assumption also made no significant difference. BODY.VIGILANCE ASSESSMENT: The reaction times from the last 3 minutes of both Go/No-Go tasks were used to evaluate vigilance. Results from two subjects were excluded, as in the Go/No-Go task. In the second Go/No-Go task, subjects whose treatment assumption after final free recall was DNK were on average 69 milliseconds slower than those who assumed placebo (P = 0.02); this was not expected. There was no significant difference between those who assumed they had received placebo after final free recall and those who assumed MPH. No significant differences were found between the real MPH and placebo groups. BODY.SUBJECTIVE RESPONSES: The POMS results showed an alteration in subjective mood the day after administration. Participants whose treatment assumption after final free recall was MPH showed significantly more anger (P = 0.031) and fatigue (P = 0.036) than those who answered placebo. This was not seen using the second set of treatment assumptions reported after 1HDFR. The VAS scale, which measured fatigue, was not affected by assumed treatment. Again, no significant differences were found between the real MPH and placebo groups. BODY.ACCURACY OF TREATMENT ASSUMPTION: Only two people (18%) who had received MPH guessed correctly after final free recall; this declined to one person (9%) after 1HDFR. Of the subjects who received a placebo, 60% guessed correctly after final free recall; this rose to 80% after 1HDFR. Subjects who received the active drug were almost three times less accurate in their guesses after final free recall than those who received the placebo, and they were six times less accurate after 1HDFR. Subjects who said they did not know what they had received were not taken into account (Table 1). BODY.POST-TEST QUESTIONNAIRE: Of the 21 subjects, 20 answered all the questions, and one subject did not respond and was excluded from the analysis. Of these 20, 14 (70%) said that they had relied on their physical signs to determine whether they had received MPH or a placebo. Two participants (10%) said they had relied on the results they expected on the immediate free recall and final free recall to guess whether or not they had taken the drug. Four (20%) said they had relied on both their physical signs and their expected results. BODY.DISCUSSION: This double-blind study was designed to explore whether the assumption that they received MPH or a placebo had an effect on cognition in healthy young adults. It explicitly investigated whether MPH and/or the assumption of having taken MPH had a facilitating effect on memory consolidation processes and reaction times under the short-term sleep deprivation conditions typically associated with an examination period. To the best of our knowledge, this is the first study that has examined the effects of MPH treatment assumptions in healthy volunteers. BODY.INTERPRETATION OF RESULTS: The effect of the treatment assumption reported by participants directly after final free recall was significant. Participants who assumed they had received MPH or who had no opinion recalled more words than those who assumed they had received a placebo. At the same time, there was no significant difference in word recall between those who really received MPH and those who did not, either on this test or on any of the other tests. Therefore, the enhanced word recall was due to differences in the subjects’ treatment assumptions, and not to the drug that they took. Several studies have shown that MPH can have an effect on mood arousal.33,34 In the present study, the assumption that they had taken MPH had a noticeable impact on subjects’ anger and fatigue levels 24 hours after administration, whereas the actual treatment taken had no effect. Participants in double-blind, randomized, placebo-controlled trials can experience treatment effects that lead to unblinding.19 For example, headaches caused by calcium channel blockers can make some participants suspect that they received the active study drug. The present study shows that assessing participants’ treatment assumptions in a randomized controlled trial can also yield important information when it is not immediately evident that participants have been unblinded by treatment effects or side effects.19 Subjects who performed more confidently on the free recall tests in this study might have assumed subsequently that they had received MPH rather than a placebo; this possibility was addressed to a certain extent by the post-test questionnaire. It might also have been valuable to ask subjects about their treatment assumptions prior to final free recall, and to assess how confident participants felt about their BODY.STUDY LIMITATIONS: Some caution is in order when interpreting the findings of this research. This study has a between-subjects design in which participants were given either a placebo or MPH. A counterbalanced within-subject design with a one-week washout period might have been preferable because it could have reduced the effect of differences between individuals. However, this was not possible due to resource constraints. Because of recruitment problems, there were only 21 volunteers in the study. Therefore, findings that were not statistically significant may have been so due to a small sample size. Tests were repeated several times using the same material, including during and immediately after the free recall study phase and several times later on. Therefore, it could be argued that the observed effects were partially caused by influences on the memory retrieval process. This was also noted by Zeeuws et al7,8,10 and Soetens et al.9 In their study, the use of immediate recall tests did not affect the influence of d-amphetamine on delayed recall.10 Because of the similar activity of MPH and d-amphetamine, it can be argued that this is likely to be true for MPH as well. One should also keep in mind that intersubject variability exists with MPH. It is rapidly and extensively absorbed after oral administration, and differences in metabolism as well as pharmacodynamic drug interactions can induce differences in bioavailability, as can nutrition effects, such as high or low fat diets and associated gastric emptying times.1,35 Subjects were not forced to choose a treatment assumption. With hindsight, it would have been better to let subjects choose between MPH, placebo, and DNK, and then force them to choose between the first two. This should probably be the case in all studies which solicit treatment assumptions. Finally, a possible explanation for the lack of difference between MPH and placebo might be that the dose was too low. Alternatively, the tests used may not be particularly good tests of MPH effects in the study population. BODY.FUTURE RESEARCH: Repeating the above study using sequential dosing of MPH or placebo would be an interesting and useful future line of research. The results of this study might lead one to conclude that students who assumed they had taken MPH during an examination period would experience more memory enhancement than those who actually took MPH. “Treatment assumption” is closely related to the placebo effect, which is thought to be associated with a spectrum of factors, including expectancy, hope, conditioning, and anxiety reduction.36 Nonblinded studies comparing placebos with no treatment options may contribute to a better appreciation of the full effect of placebos.37 Additional research with a larger study population is needed to elucidate better the precise nature of “treatment assumption”. Future trials with longer follow-up and larger study populations may also clarify the effects of MPH treatment assumption in healthy subjects, especially when combined with an understanding of subjects’ prior expectations about treatment efficacy. BODY.CONCLUSION: This study showed that the assumption of having received a single 20 mg dose of MPH may induce a substantial placebo effect in healthy young students, improving consolidation of information into long-term memory. Students who thought they were given the drug recalled 54%–58% of the word lists, while those who thought they had received the placebo recalled only 34%–37%. This is independent of any pharmacologic effects of MPH, which in this study had no significant effects on verbal memory. This information may be used to dissuade students from taking stimulants such as MPH during their examination periods, thus avoiding one trap towards becoming addicted subsequently.38,39 In addition to this, pointing towards the placebo effect indicates to students that they have the psychologic means within themselves to enhance their own study capacity without any need for drugs.

TITLE: Insulin treatment guided by subcutaneous continuous glucose monitoring compared to frequent point-of-care measurement in critically ill patients: a randomized controlled trial ABSTRACT.INTRODUCTION: Glucose measurement in intensive care medicine is performed intermittently with the risk of undetected hypoglycemia. The workload for the ICU nursing staff is substantial. Subcutaneous continuous glucose monitoring (CGM) systems are available and may be able to solve some of these issues in critically ill patients. ABSTRACT.METHODS: In a randomized controlled design in a mixed ICU in a teaching hospital we compared the use of subcutaneous CGM with frequent point of care (POC) to guide insulin treatment. Adult critically ill patients with an expected stay of more than 24 hours and in need of insulin therapy were included. All patients received subcutaneous CGM. CGM data were blinded in the control group, whereas in the intervention group these data were used to feed a computerized glucose regulation algorithm. The same algorithm was used in the control group fed by intermittent POC glucose measurements. Safety was assessed with the incidence of severe hypoglycemia (<2.2 mmol/L), efficacy with the percentage time in target range (5.0 to 9.0 mmol/L). In addition, we assessed nursing workload and costs. ABSTRACT.RESULTS: In this study, 87 patients were randomized to the intervention and 90 to the control group. CGM device failure resulted in 78 and 78 patients for analysis. The incidence of severe glycemia and percentage of time within target range was similar in both groups. A significant reduction in daily nursing workload for glucose control was found in the intervention group (17 versus 36 minutes; P <0.001). Mean daily costs per patient were significantly reduced with EUR 12 (95% CI −32 to −18, P = 0.02) in the intervention group. ABSTRACT.CONCLUSIONS: Subcutaneous CGM to guide insulin treatment in critically ill patients is as safe and effective as intermittent point-of-care measurements and reduces nursing workload and daily costs. A new algorithm designed for frequent measurements may lead to improved performance and should precede clinical implementation. ABSTRACT.TRIAL REGISTRATION: Clinicaltrials.gov, NCT01526044. Registered 1 February 2012. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13054-014-0453-9) contains supplementary material, which is available to authorized users. BODY.INTRODUCTION: Stress-induced hyperglycemia is common and relates to adverse outcomes in critically ill patients [1,2]. The outcomes of two large intervention studies are in some way contradictory but the consensus is that hyperglycemia should be corrected, while avoiding hypoglycemia and high glucose variability [3-8]. On the basis of the available evidence, it seems preferable to maintain a blood glucose level around 8.0 mmol/L for the majority of critically ill patients [9,10]. Glucose regulation regimens require frequent monitoring of glucose, which leads to a considerable workload for the intensive care (IC) nurses. In addition, glucose regulation carries an inherent risk of insulin-induced hypoglycemia, which is associated with mortality [6]. Information about the glucose level is lacking for the period in between measurements with possible unnoticed hypoglycemic episodes. Continuous glucose monitoring (CGM) could be of value to facilitate or improve glycemic control. Previous studies have indicated an acceptable accuracy and reliability for subcutaneous CGM devices in critically ill patients [11-15]. The only prospective randomized controlled trial so far that assessed the role for CGM in glycemic control in critically ill patients showed that real-time CGM increased the safety of tight glycemic control in critically ill patients by significantly reducing severe hypoglycemic events [16]. However, an improvement of the mean glucose concentration by using real-time CGM was not found [16]. Thus, CGM may give us the ability to detect early (possible) hypo- and hyperglycemia as well as minimizing swings in glucose levels. Moreover, the use of CGM may facilitate the process of glycemic control and may reduce the number of blood samples and accompanying blood loss, nursing workload and costs. To date, there are few data available how CGM-driven glucose regulation compares to point-of-care (POC) -driven glucose regulation and no controlled studies specifically evaluated workload and cost of CGM. The aim of the present study was to assess the safety, efficacy, workload and costs of a subcutaneous CGM system-guided blood glucose regulation in comparison with frequent POC blood glucose-guided regulation in a mixed population of critically ill patients. BODY.MATERIAL AND METHODS: BODY.STUDY DESIGN AND PARTICIPANTS: This was a randomized controlled open-label clinical trial, performed in a 20-bed mixed medical-surgical ICU of a teaching hospital (Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands). Patients were recruited over a period of 18 months from 2011 till late 2012. Patients were eligible for inclusion within 24 hours after ICU admission if they were 18 years or older, in need of intravenous (i.v.) insulin treatment for glucose regulation and with an expected length of stay in the ICU of at least 24 hours. Patients could not be included if any of the following criteria was present: lack of informed consent, participation in another trial or previous participation in this trial or when a CGM system was currently not available. The study ended when patients were discharged from the ICU or because of technical failure of the CGM device. The maximum study duration was set at five days for both treatment groups. The complete nursing staff was trained beforehand to handle all devices used in this study adequately. This study was approved by the ethics committee VCMO, Nieuwegein, The Netherlands and was in line with Dutch and European legislation. All patients or their legal representative provided written informed consent. This trial is registered with Clinicaltrials.gov, number NCT01526044. BODY.RANDOMIZATION: Patients who met the inclusion criteria were randomized in a 1:1 ratio with computerized block randomization to either the intervention group or the control group. BODY.STUDY PROCEDURES: BODY.ALGORITHM: In all study participants, blood glucose regulation was performed by a sliding scale algorithm with a blood glucose target of 5.0 to 9.0 mmol/L, which was integrated into the patient data management system (PDMS, MetaVision; iMDsoft, Tel Aviv, Israel) [17]. Hypoglycemia was defined as a blood glucose level of <2.2 mmol/L in line with the Van den Berghe trial [3]. Below target was defined as a glucose level from 2.2 mmol/L till the lower target level of 5.0 mmol/L. Above target, all glucose levels were above 9.0 mmol/L. The algorithm instructed the insulin i.v. infusion rate (or glucose administration in case of hypoglycemia) after each glucose measurement. The time for the next glucose measurement was also defined from the algorithm and depended on the stability of the glucose level over time. BODY.GLUCOSE MEASUREMENT: Study participants allocated to the intervention group received a subcutaneous CGM system (FreeStyle Navigator™, Abbott Diabetes Care, Alameda, CA, USA), which was used to guide blood glucose regulation. The nurses were trained to insert the subcutaneous glucose sensors on the patients’ abdomen or upper arm. After insertion of the subcutaneous sensor, a transmitter was attached that connects through wireless communication to a receiver, which displays the real-time glucose readings every minute and stores glucose readings every 10th minute. The CGM system needed a one-hour stabilization period, in which glucose measurements were not performed. Calibration of the system using an arterial blood sample was performed five times in total, after 1, 2, 8 to 10, 24 to 32 and 72 to 80 hours, following manufacturer instructions. The CGM system sounded an alarm when additional calibrations were needed. On the times that the algorithm needed a new glucose measurement, the readings from the CGM system were entered in the computerized glucose regulation protocol that was embedded in the PDMS. Other CGM values were not used in the algorithm. The CGM system sounded an alarm when the glucose level was either <5.0 mmol/L or >9.0 mmol/L. When this occurred, the nurse entered this additional glucose level in the computerized protocol, which triggered the glucose algorithm to advise an insulin dosing adjustment. The CGM repeated its alarm after 15 minutes when the glucose level was still out of target range. Again, this value was entered into the system and dose adjustments were made until target range was achieved. Every hypoglycemic event (<2.2 mmol/L) needed to be verified by an arterial blood glucose sample. In case of a discrepancy between the CGM value and the arterial blood glucose sample, the latter was leading in clinical decision-making. Blood glucose regulation in the study participants allocated to the control group was performed by use of frequent point-of-care (POC) measurements using Accu-Chek™ (Roche/Hitachi, Basel, Switzerland). All blood samples were obtained from an indwelling arterial catheter. The displayed glucose levels were automatically stored in the PDMS. Participants in the control group also received a subcutaneous Freestyle Navigator CGM system, however, these data were blinded and not used for blood glucose regulation. Calibrations were performed following manufacturer instructions and no alarms were set. In both groups arterial reference blood glucose samples were drawn six times daily at standardized times and analyzed by the ABL Flex automated blood gas analyzer (BGA) (Radiometer, Copenhagen, Denmark). These values were automatically stored into the PDMS but were blinded to both nurses and physicians. BODY.STUDY ENDPOINTS: The primary safety outcome was the incidence of severe hypoglycemia (<2.2 mmol/L) during the intervention. Efficacy outcomes were the percentage of time that glucose levels were within the target range (5.0 to 9.0 mmol/L), below target range (2.2 to 5.0 mmol/L), and in the hyperglycemic range (>9.0 mmol/L). In addition, mean blood and sensor glucose levels and glucose variability defined as the mean absolute glucose (MAG) change (ΔGlucose/ΔTime) were endpoints too [8]. The accuracy of the CGM and the POC device was assessed by calculating the median relative absolute deviation (RAD) between reference glucose and CGM or POC glucose. Nursing workload for glucose control per day was determined by the number of POC measurements or measurements from the sensor, which were entered in the computerized glucose regulation protocol and the amount of calibrations of the CGM sensor (in the intervention group only). A time-in-motion design was used to estimate the time that it took to execute targeted glucose control and insulin treatment per group. The following subtasks were observed: (1) POC measurement (this included the initiation, blood sampling, blood testing and processing), (2) sensor placement, (3) sensor calibration and (4) time needed to determine a CGM value and entering the value in the decision support module. The tenfold-recorded elapsed times per subtask were averaged and then multiplied by the 24-hour blood sample average collected from the clinical trial. Cost analysis was performed from a health-care payer perspective with a one-day (24 hours) time horizon. The outcome measure in the economic evaluation was the costs per patient for glycemic control in 24 hours. Cost parameters included nursing personnel costs, device costs, materials needed for glucose monitoring and laboratory costs. Cost estimates for the parameters were derived from the hospital and laboratory ledger, devices manufacturers’ data and the Dutch guide for health economic research [18]. Costs are expressed in euros and are based on the year 2013. Because of the short time horizon of this analysis (24 hours), the costs were not discounted. BODY.DATA COLLECTION: Clinical and laboratory baseline data were extracted from the PDMS after randomization: demographic data, body mass index (BMI), reason for ICU admission, history of diabetes, history of renal failure, severity of disease scores (the sequential organ failure assessment (SOFA) score and acute physiology and chronic health evaluation (APACHE IV) score at admission), blood glucose levels at admission and the use of mechanical ventilation. Blood glucose data, that is reference arterial blood glucose samples and glucose values that were entered in the decision support module (CGM measurements in the intervention group, POC measurements in the control group) were also extracted from the PDMS. Continuous glucose data from the CGM device were uploaded to a computer using CoPilot™ Health Management System for FreeStyle Navigator (Abbott Diabetes Care, Alameda, CA, USA) and entered in the study database. All reference glucose measurements were linked by time with the concomitant CGM measurements and Accu-Chek measurements. BODY.STATISTICAL ANALYSIS: A sample size of 160 (80 participants in each group) conferred 80% power, with two-sided P = 0.05, to detect an absolute difference of 10% in the incidence of severe hypo- or hyperglycemia between the intervention and the control group. A total sample size of 178 patients (89 patients per group) is needed to correct for an expected 10% drop out. Results are expressed as percentages for categorical variables, mean and standard deviation (SD) for continuous normally distributed variables, and median and interquartile range (IQR) for continuous non-normally distributed variables. Groups were compared by using Fisher’s exact test, Student’s t test or Mann-Whitney rank-sum test as appropriate. Median RAD was calculated instead of mean because of its skewed distribution. Costs were calculated as the summed product of factors and resources used and their respective unit costs and were averaged per patient per day. Because of skewed (cost) distributions, we assessed group contrasts by calculating 95% confidence intervals for the mean differences following bias-corrected and accelerated nonparametric bootstrapping, that is drawing 1,000 samples of the same size as the original sample separately for each group. All statistical analyses were performed in SPSS 20.0 (IBM Corp, Armonk, NY, USA). BODY.RESULTS: A total of 178 patients were randomized to either the intervention or the control group (Figure 1). Most of the patients who were not eligible were postoperative cardiac surgery patients with an expected length of stay (LOS) <24 hours. One patient was incorrectly randomized and did not receive a CGM device. Nine patients in the intervention group and twelve patients in the control group were excluded from analysis due to lack of CGM data because of technical failure of the device, misplacement of the sensor (n = 3) and problems with extraction of the data (n = 18).Figure 1 Flow chart of study participants: assessment, randomization and analysis. We performed a per protocol analysis from the data of 78 patients in each group. Table 1 shows the two groups, which were well matched with respect to all baseline characteristics.Table 1 Baseline characteristics of participants Intervention - CGM Control - POCM ( n  = 87) ( n  = 90) Age (years) 664 (14.0) 67.2 (11.4) Women 45 (52%) 35 (39%) BMI (kg/m2) 27.8 (7.0) 27.4 (5.8) Weight (kg) 81.8 (21.7) 83.2 (21.5) History of diabetes* 18 (21%) 21 (23%) History of renal failure** 10 (12%) 5 (6%) Reason for ICU admission Surgical  Elective 19 (22%) 16 (18%)  Emergency 12 (14%) 13 (14%) Medical 56 (64%) 61 (68%) Admission diagnosis Post cardiac surgery 12 (14%) 11 (12%) Severe sepsis/septic shock 23 (26%) 18 (20%) Pneumonia 12 (14%) 11 (12%) Cardiac failure 10 (12%) 9 (10%) COPD 3 (3%) 8 (9%) Hemorrhagic shock 7 (8%) 10 (11%) Cardiac arrest/resuscitation 10 (12%) 14(16%) Other 10 (12%) 9 (10%) APACHE IV predicted mortality (%) 32 (10-70) 31 (20-60) SOFA score on admission 8 (6-10) 7 (6-10) Blood glucose level on admission (mmol/L) 9.0 (2.6) 9.2 (2.5) Mechanical ventilation 80 (92%) 83 (92%) Data are mean (SD), median (IQR) or n (%). *Diabetes was defined as present when this diagnosis was mentioned in the medical history; **renal failure was present when the preadmission serum creatinine was above 177umol/l. CGM: continuous glucose monitoring, POCM: point-of-care monitoring, BMI: body mass index, ICU: intensive care unit, SOFA: sequential organ failure assessment, COPD: chronic obstructive pulmonary disease; APACHE: acute physiology and chronic health evaluation. During the intervention, a total of 37,570 (intervention group) and 32,957 (control group) CGM measurements were collected. The number of reference arterial blood gas glucose measurements was 1,599 in the intervention group and 1,325 in the control group. The median number of additional calibrations needed for the CGM was 1.9 per 24 hours (IQR 1.2 to 3.3). The number of glucose values entered in the PDMS (CGM measurements in the intervention group and POC measurements in the control group) was 3,919 and 2,489 respectively. Table 2 summarizes the outcome measures of the study. The incidence of hypoglycemia (<2.2 mmol/L), the primary safety endpoint, was similar in both the intervention and the control group. None of the severe hypoglycemic episodes detected by the CGM in the intervention group was verified by arterial blood sampling. In the control group, all severe hypoglycemic episodes detected by the CGM, occurred in between two POC glucose measurements and were not detected by the nurses. In total, there were 14 patients (3 patients in the control group and 11 patients in the intervention group) who experienced 19 ‘true’ hypoglycemic events (<3.9 mmol/L) detected by ABL. Twenty-five percent (n = 4) of the true ‘hypoglycemic’ events in the CGM group and 67% (n = 2) in the control group were also identified by CGM or POC (difference in glucose ≤10%). All other endpoints such as percentage time in target range, below target range, mean reference and sensor glucose, glucose variability, hospital LOS, ICU and hospital mortality were nonsignificantly different between the study groups. Moderate hyperglycemia (9.0 to 11.1 mmol/L) was significantly different in favor of the intervention group (P = 0.03). A total of 355 time-linked reference glucose CGM samples and 85 time-linked reference glucose POC samples were used to assess accuracy of the devices. Median (IQR) RAD of the POC device was 7.1% (3 to 12) whereas the median RAD of the CGM device was 13.7% (8 to 23) (P <0.001). Bland-Altman plots per glucose monitoring system are shown in an additional file (Figure S1 in Additional file 1).Table 2 Safety, efficacy and clinical study outcomes Intervention - CGM Control - POCM P ( n  = 78) ( n  = 78) Study period (days) 3.2 (2-5) 2.8 (1-5) 0.18 Incidence severe hypoglycemia (<2.2 mmol/L)1 None None Detected by CGM  Number of subjects 3 (4%) 4 (5%) 1.0  Episodes < 2.2 mmol/L 3 4 % of time for the reference glucose level (SD)3 In target range (5.0-9.0 mmol/L) 69 (26) 66 (26) 0.47 Below target range (2.2-5.0 mmol/L) 5 (7) 3 (5) 0.21  Mild/moderate hypoglycemia (2.2-3.9) 1 (3) 0 (1) 0.03 Above target range (>9.0 mmol/L) 28 (26) 34 (27) 0.06  Mild/moderate hyperglycemia (9.0-11.1) 17 (16) 26 (23) 0.01  Hyperglycemia (>11.1) 11(19) 7(14) 0.19 % of time for the sensor glucose levels (SD)3 In target range (5.0-9.0 mmol/L) 75 (18) 71 (20) 0.18 Below target range (2.2-5.0 mmol/L) 11 (13) 9 (12) 0.44  Mild/moderate hypoglycemia (2.2-3.9) 2 (7) 1 (2) 0.14 Above target range (>9.0 mmol/L) 15 (16) 20 (21) 0.06  Mild/moderate hyperglycemia (9.0-11.1) 12 (11) 16 (16) 0.03  Hyperglycemia (>11.1) 3 (7) 4 (9) 0.35 Mean reference blood glucose (mmol/L) 8.2 (1.6) 8.3 (1.3) 0.53 Mean sensor glucose (mmol/L) 7.1 (1.1) 7.5 (1.3) 0.07 MAG change (mmol/L/h)2 0.33 (0.2-0.5) 0.32(0.2-0.4) 0.31 LOS ICU (hours) 137 (71-250) 95 (51-157) 0.04 LOS hospital (days) 15 (8-270) 14 (8-31) 0.91 Mortality ICU 15 (19%) 12 (15%) 0.67 Mortality hospital 22 (28%) 17 (22%) 0.46 Data shown are mean (SD), median (IQR), or n (%). 1Patients who experienced at least one severe hypo- or hyperglycemic episode, verified by blood gas analysis; 2when at least three reference glucose measurements were available (intervention n = 73, control n = 71); 3percentages do not add up to 100 due to rounding off. CGM: continuous glucose monitoring; POCM: point-of-care measurement, MAG: mean absolute glucose change; LOS: length of stay; ICU: intensive care unit. Table 3 summarizes nursing workload data per 24 hours. The first column displays the average time burden per subtask of glucose control. The average total time burden for glucose control was significantly lower in the intervention group compared to the control group (17 minutes versus 36 minutes; P <0.001). The mean reduction in total nursing workload was 19 minutes per 24 hours or 53% in favour of the intervention group. As in this study, an open blood drawing system was used, 5 mL blood per POC measurement or calibration was taken from the patient. Blood loss was therefore significantly reduced in the intervention group (15.3 mL versus 60 mL per day; P <0.001).Table 3 Nursing workload per day (24 hours) Time per action (min) Number of actions in control group Nursing time control group (min) Number of actions in intervention group Nursing time intervention group (min) POC measurement 3 12 (8) 36 (24) 0.06 (0.4) 0.2 (0.4) Sensor CGM placement 3.5 - - 1 3.5 Sensor CGM calibration 2.5 - - 1.9 (1.2-3.3) 8 (11) Sensor CGM data to enter in PDMS 0.3 - - 18 (10) 5.3 (3) Total time (min) 36 (24) 17 (12)* Data are expressed as mean (SD), or median (IQR). *P <0.001 in comparison with control group. POC: point-of-care; CGM: continuous glucose monitoring; PDMS: patient data management system. The economic analysis of both groups is shown in Table 4. The intervention group generated an average total daily cost of EUR 41, whereas the total daily cost in the control group was EUR 53. The difference in costs was EUR −12 in favor of the intervention group (95% CI −32 to −18, P = 0.02). The extra costs of the CGM devices in the intervention group were neutralized by the diminished costs for nursing personnel, material and laboratory costs.Table 4 Cost analysis Costs per unit Factor control group Costs in control group Factor intervention group Costs in intervention group Difference in costs (95% CI) 1 Nursing time €38/hr 36 min €22.98 17 min €10.87 €-12.11(−16, −9) CGM receiver €1009.59 - - €1.38 per day2 €1.38 €1.38 CGM sensor €61.00 - - €24.40 per day3 €24.40 €24.40 CGM calibration4 €1.19 - - 3.3 €3.95 €3.95 (3,5) Accu-Chek Inform II device €892.37 €1.22 per day2 €1.22 - - €-1.22 Material POC measurement5 €0.70 12.2 €8.51 0.06 €0.04 €-8.47 (−10, −7) Laboratory6 €1.66 12.2 €20.18 0.06 €0.10 €-20.08 (−23, −18) Total costs €52.89 €40.74 €-12.42 (−22, −5) Factors and costs are expressed as means per patient per day (24 hours). 195% confidence interval based on 1,000 stratified bootstrap samples; 2assuming a lifetime of two years; 3assuming a manufacturers’ sensor lifetime of two and a half days; 4calibration strip CGM; 5includes syringes, nonsterile gloves, gauzes, alcohol, cap (used for blood sampling) and testing strip POC; 6costs for a single point-of-care glucose measurement. CGM: continuous glucose monitoring, POC: point-of-care. BODY.DISCUSSION: The present study showed that a subcutaneous CGM system to guide blood glucose regulation was equally effective and safe in glycemic control compared to frequent POC-guided blood glucose regulation. However, CGM significantly reduces nursing workload, blood loss and the daily costs for glucose control. BODY.COMPARISON WITH OTHER STUDIES: This is the second but largest randomized controlled trial in which CGM is used to guide glycemic control in critically ill patients. In contrast to our findings, Holzinger and colleagues did find less severe hypoglycemia in the CGM group [16]. This may be caused by the very low incidence of severe hypoglycemia in the present study, which was true for both the intervention and the control group. This may be related to a change of policy after the publication of the NICE-SUGAR trial [4], which was a reason for our and most other ICUs to increase their blood glucose target range. The increased target range may have reduced the incidence of hypoglycemic events [19,20]. Indeed, the blood glucose target used in the current study (5.0 to 9.0 mmol/L) was higher than in the Holzinger trial [16] (4.4 to 6.1 mmol/L) and this is reflected in the achieved mean blood glucose levels (8.1 vs. 6.3 mmol/L). Moreover, the use of a fully computerized algorithm for glucose control and the high familiarity of the protocol among our IC nurses may have contributed to the low incidence of severe hypoglycemia. The available studies to date on tight glucose control showed an increase in nursing workload [21-23]. The potential benefits of CGM in the reduction of blood samples, blood loss and nursing workload was assumed in previous studies, but was not systematically assessed before. We now observed that CGM significantly reduced the amount of blood samples and the daily nursing workload for glucose control up to 53%. This finding seems clinically relevant, especially in a busy clinical IC environment. Two studies focused on the cumulative nursing workload accompanied with tight glucose control protocols [21,22]. Gartemann et al. estimated that nurses devoted approximately 42 minutes during a 12-hour shift of their time to administering a tight glycemic control (TGC) protocol, whereas Aragon et al. even reported that up to 2 hours might be required for tight glycemic control for a single patient in a 24-hour period. In our POC control group, the mean nursing workload estimate was less (36 minutes per 24 hours) than the published estimates reported by other groups. This might partly be explained by the use of a fully computerized algorithm for glucose control in our ICU. In addition, the familiarity of the protocol is very high among our ICU nurses. BODY.EFFECTIVENESS AND COSTS: The use of CGM did not achieve improved glycemic control in our study. We found similar percentages of time-in-target and below-target range between the study groups. The not-significantly lower percentage of time in the hyperglycemic range in the intervention group could be explained by the fact that CGM measurements were more frequently entered in the glucose protocol than POC measurements in the control group. This probably resulted in more adjustments in the insulin treatment with lower blood glucose levels as a consequence. The significantly increased ICU LOS, which was observed in the intervention group, may be a coincidence or reflect unmeasured case-mix factors but is, in our view, unrelated to the glucose measurement strategy. In contrast to our expectations, the cost analysis shows that the use of CGM systems for glucose control in an ICU setting is not a priori an expense. However, we should be cautious in interpreting these results due to the rather short time horizon (24 hours) in the analysis of costs determination and the single-centre study design. Also, cost savings cannot immediately be monetized due to the short time horizon used in this cost analysis. BODY.ACCURACY OF THE SUBCUTANEOUS MEASUREMENTS: The subcutaneous Freestyle Navigator CGM device that we used in the present study showed a median RAD of 13.7%, which is higher than the 10.6 and 11.6% that was found in previous validation studies of this device in critically ill patients, suggesting an accuracy acceptable for clinical use. [11,14]. The lag time that may be needed for the subcutaneous compartment to adapt to the intravenous compartment appeared not to be clinically relevant [11]. However, the accuracy as assessed in the current study seems to indicate a need for improvement, because the accuracy was less than the accuracy of the Accu-Chek and because a substantial number (75% in the CGM group and 33% in the control group) of hypoglycemic events was not detected. Of note, Leelarathna et al. [24] recently investigated whether there was a difference in accuracy of the Freestyle Navigator in a critical care setting using two methods of calibration: (1) calibration according to the manufacturer’s instructions (1, 2, 10, and 24 h) or (2) calibration at variable intervals of 1 to 6 h using ABG. Using enhanced calibration, at a median (interquartile range) every 169 (122 to 213) minutes, the absolute relative deviation was lower (7.0% (3.5, 13.0) vs. 12.8% (6.3, 21.8), P <0.001). So, further significant improvements in accuracy may be obtained by frequent calibrations with ABG measurements. In the current study forced calibration was not possible, calibration was only performed when the CGM device indicated the need for calibration by itself. In addition, technical problems with the subcutaneous CGM device were observed during the study and led to a 12% dropout. The most important reason was the temporary loss of sensor signal from several minutes to hours that resulted in a loss of data. Difficulties in the calibration process were also identified as the CGM could only be calibrated if the system indicated a calibration by itself, which occurred for median 1.9 times per 24 hours. Most of the technical difficulties, however, may have been due to lack of experience working with the CGM device despite the training of all ICU nurses. We expect such problems to be easily resolved with additional training and with the improved next generation Freestyle Navigator II, which has recently been introduced and showed good utility and sensor performance in critically ill patients [25]. This study aimed to define safety, efficacy and costs and therefore we neglected the system dropout at this moment. It is true, however, that this device can only become part of routine care when the dropout percentage diminishes. BODY.STRENGTHS AND WEAKNESSES: The strengths of our study include the relatively large sample size, the randomised controlled study design and the wide variety in case mix. However, some limitations of the present study merit further consideration. First, the study was performed in a single Dutch intensive care unit, which limits the generalizability of the study. Second, the study was designed to blind the values of the CGM in the control group. However, the CGM needed to be calibrated several times during the study period, which made it impossible to blind it completely. Third, the nursing staff did not verify the severe hypoglycemia that was indicated by CGM in two of the three patients despite specific instructions to do so. One of these two patients had evolved into a ‘withholding care policy’, which was the reason to accept the severe hypoglycemia. We assume that in the other patient priority was given to other important nursing tasks. Thus, the available data are insufficient to define the accuracy of the CGM in the hypoglycemic range. In our previous studies this was not identified as a clinical problem [11,14]. Also, with an adapted algorithm, the CGM should be able to detect a decreasing glucose level before hypoglycemia is present and give a timely alert. Fourth, the computerized algorithm was designed for intermittent POC measurements and not for (semi-) continuous data. As such, the patients did not fully benefit from the frequent glucose measurements by CGM. An algorithm based on 10-minute glucose input might have led to other results. We did identify this issue beforehand but we decided to keep the algorithm for both groups the same to be able to investigate the contribution of CGM per se. It can be expected that an adapted algorithm will further improve the performance of CGM in the guidance of glycemic control. BODY.CONCLUSIONS: Subcutaneous CGM to guide blood glucose regulation in critically ill patients was shown to be safe in terms of hypoglycemia incidence. With an identical insulin treatment algorithm, the CGM was equally effective as POC measurement. A new algorithm designed for frequent measurements may further improve the results and should precede clinical implementation. CGM significantly reduced nursing workload, blood loss and the daily costs for glucose control. BODY.KEY MESSAGES: Insulin treatment based on continuous subcutaneous glucose monitoring (CGM) revealed the same number of hypoglycemic events compared to point of care (POC) Subcutaneous CGM was equally effective as POC measured as glucose time in target range Total costs were lower when using subcutaneous CGM than frequent POC Nursing workload with glucose regulation was reduced by subcutaneous CGM compared to frequent POC A new algorithm designed for continuous measurement should be developed before CGM can be implemented clinically

TITLE: Safety and efficacy of tiotropium in patients switching from HandiHaler to Respimat in the TIOSPIR trial ABSTRACT.OBJECTIVES: This post hoc analysis of TIOtropium Safety and Performance In Respimat (TIOSPIR) evaluated safety and exacerbation efficacy in patients with stable (≥2 months) use of tiotropium HandiHaler 18 µg (HH18) prior to study entry, to evaluate whether there was a difference in risk for patients who switched from HH18 to tiotropium Respimat 2.5 µg (R2.5) or 5 µg (R5). ABSTRACT.SETTING: TIOSPIR (n=17 135) was an international, Phase IIIb/IV, randomised, double-blind, parallel-group, event-driven trial. ABSTRACT.PARTICIPANTS: Patients from TIOSPIR with chronic obstructive pulmonary disease (COPD) and postbronchodilator ratio of forced expiratory volume in 1 s to forced vital capacity ≤0.70, receiving HH18 before study entry, were analysed (n=2784). ABSTRACT.INTERVENTIONS: Patients were randomised to once-daily tiotropium R2.5 (n=914), R5 (n=918) or HH18 (n=952) for 2–3 years. ABSTRACT.PRIMARY AND SECONDARY OUTCOME MEASURES: Primary outcomes: time to death (safety) and time to first COPD exacerbation (efficacy). Secondary outcomes: number of exacerbations and time to first major adverse cardiovascular event (MACE). ABSTRACT.RESULTS: Baseline characteristics were similar in all groups. Respimat had a similar mortality risk versus HH18 (vital status follow-up, HR; 95% CI R2.5: 0.87; 0.64 to 1.17; R5: 0.79; 0.58 to 1.07) with no significant differences in the risk and rates of exacerbations and severe exacerbations across treatment groups. Risk of MACE and fatal MACE was similar for Respimat versus HH18 (HR; 95% CI MACE R2.5: 0.73; 0.47 to 1.15; R5: 0.69; 0.44 to 1.08; fatal MACE R2.5: 0.57; 0.27 to 1.19; R5: 0.67; 0.33 to 1.34). Overall risk of a fatal event (on treatment) was lower for R5 versus HH18 (HR; 95% CI R2.5: 0.78; 0.55 to 1.09; R5: 0.62; 0.43 to 0.89). ABSTRACT.CONCLUSIONS: This analysis indicates that it is safe to switch patients from tiotropium HandiHaler to tiotropium Respimat, and that the efficacy is maintained over the switch. ABSTRACT.TRIAL REGISTRATION NUMBER: NCT01126437; Post-results. BODY.STRENGTHS AND LIMITATIONS OF THIS STUDY: This post hoc analysis examined whether there was a difference in mortality or efficacy between those patients who were previously stable users (≥2 months) of tiotropium HandiHaler and continued with this delivery system during TIOSPIR and those who were subsequently randomised to tiotropium Respimat. This study will therefore be informative for physicians who are contemplating switching their patients from one inhaler to another. Being one of the largest, long-term, randomised trials of a broad range of patients with chronic obstructive pulmonary disease (COPD) conducted to date, TIOSPIR lends itself to statistically powerful subgroup analyses. The inclusion of a wide range of spirometrically defined disease severities and the liberal inclusion criteria permitted a patient population that was highly representative of a typical COPD population (including patients with cardiac disorders). The results of this study are more robust than findings from meta-analyses or database studies, which typically evaluate less frequent outcomes or rare events that may not be as rigorously collected as primary or secondary events, and in which confounding factors (such as disease severity) may not be fully adjusted for. A key limitation of the study is that it was a post hoc rather than predefined subgroup analysis. In addition, the design of the analysis does not allow for an evaluation of treatment initiation with the two inhalers in anticholinergic-naïve patients (results for this patient group are the subject of a separate analysis). A further limitation of the study is that information on duration of treatment with tiotropium HandiHaler prior to randomisation and switch to Respimat was not collected. These data would provide additional valuable information on the characteristics of the patients enrolled in the study (eg, the proportion of patients who may have been considered more ‘stable’ on treatment with tiotropium due to a longer duration of therapy than other patients). The current analysis also excluded patients with moderate-to-severe renal impairment; however, placebo-controlled studies of patients with moderate renal impairment do not indicate an increased risk in these patients with the use of tiotropium Respimat. BODY.INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is a major cause of death and disability worldwide.1 Tiotropium bromide is a once-daily, long-acting anticholinergic (long-acting muscarinic antagonist (LAMA)) and an established treatment option for COPD (SPIRIVA, Boehringer Ingelheim, Ingelheim, Germany). It is available in two formulations: a dry powder delivered via the HandiHaler device (18 µg once daily) and an aqueous solution delivered via the Respimat Soft Mist Inhaler (5 µg once daily).2 3 Tiotropium HandiHaler and Respimat have demonstrated similar improvements in lung function, symptoms and quality of life; they have similar pharmacokinetic profiles in patients with COPD.4–8 There is a large body of evidence supporting the clinical efficacy and safety of tiotropium administered via HandiHaler in patients with COPD.6 Safety concerns, however, were prompted after results of a post hoc pooled analysis of several trials (and subsequent meta-analyses) indicated a signal of increased risk of mortality with tiotropium Respimat, notably in patients with a history of cardiac arrhythmias.9–14 A pooled analysis of tiotropium HandiHaler studies, as well as the 4-year Understanding Potential Long-term Impacts on Function with Tiotropium (UPLIFT) (NCT00144339) study,8 15 16 did not indicate such a risk, with rate ratios favouring tiotropium HandiHaler versus placebo. Nevertheless, a possible interpretation of data from additional studies14 17 could have suggested a specific risk associated with tiotropium Respimat. The TIOtropium Safety and Performance In Respimat (TIOSPIR) (NCT01126437) trial18 19 was conducted to provide a prospective evaluation of the safety and exacerbation efficacy of tiotropium Respimat versus tiotropium HandiHaler. TIOSPIR is the largest long-term, randomised, double-blind study in patients with COPD performed to date. It demonstrated that the risk of death was similar for tiotropium Respimat, at a dose of 2.5 or 5 µg, to that of HandiHaler 18 µg, with a HR of 0.96 for tiotropium Respimat 5 µg versus HandiHaler 18 µg. The risk of exacerbation and major adverse cardiovascular (CV) events (MACEs) did not differ significantly among the three groups.19 Despite providing reassurance on the safety of Respimat versus HandiHaler, the results from TIOSPIR have continued to raise some important questions regarding the two formulations. In particular, a population cohort study of patients aged ≥45 years receiving tiotropium identified several factors that influenced the likelihood of switching from HandiHaler to Respimat.20 These included COPD severity and comorbidities (notably neurological conditions).20 The authors of the study concluded that tiotropium users in clinical practice typically have more severe disease than patients included in randomised clinical trials and, therefore, called for further studies comparing the safety profile of the two tiotropium formulations.20 This post hoc analysis of TIOSPIR was conducted to address these concerns and to assess whether there was a difference in safety or efficacy between patients who had already received stable treatment with tiotropium HandiHaler and continued with this delivery system, and those who were subsequently randomised to tiotropium Respimat. BODY.METHODS: BODY.STUDY DESIGN: TIOSPIR was a long-term (2–3 years), international, multicentre, Phase IIIb, randomised, double-blind, double-dummy, parallel-group, actively controlled, event-driven trial in a large population (n=17 135) of patients with COPD. The study design has been described previously.18 Patients were randomised to one of three treatments: once daily tiotropium Respimat 2.5 μg (two puffs of 1.25 µg) or 5 µg (two puffs of 2.5 µg) or once daily tiotropium HandiHaler 18 μg. The study did not include a washout period from COPD treatments prior to randomisation. Patients attended clinic visits every 12 weeks, with a final visit 30 days after the end of treatment; all were followed up for vital status until the end of the study, even if their treatment was discontinued. The trial was performed in accordance with the Declaration of Helsinki, and the study protocol and procedures were approved by the relevant institutional review boards and ethics committees. All patients provided written informed consent. BODY.STUDY POPULATION: Tiotropium HandiHaler 18 μg was the only LAMA marketed (tiotropium Respimat was not available) at the time of study enrolment (May 2010–April 2011) in the following countries: Australia, Canada, China, Georgia, Guatemala, India, Israel, New Zealand, Switzerland, Thailand, Tunisia and the USA. This subgroup analysis included patients with stable LAMA use prior to study enrolment (in the previous 2 months prior to visit 1) who were recruited from 339 centres across these countries, resulting in a population restricted to patients using tiotropium HandiHaler 18 μg. Patients were aged ≥40 years with a clinical diagnosis of COPD, postbronchodilator ratio of forced expiratory volume in 1 s (FEV1) to forced vital capacity ≤0.70, FEV1≤70% predicted, and had ≥10 pack-years of smoking history. Exclusion criteria were chosen to select a typical COPD population. Patients with concomitant cardiac disease were permitted to participate in the study, unless they had experienced a myocardial infarction (MI) within the previous 6 months, were hospitalised for New York Heart Association class III or IV heart failure, or had a history of unstable or life-threatening arrhythmia requiring new treatment within the previous 12 months. In addition, patients with unstable COPD (exacerbation within 4 weeks), moderate or severe renal impairment as judged by the investigator, or other significant lung diseases, were excluded. Patients were permitted to use their usual background treatment for COPD, except for other inhaled anticholinergics. BODY.ASSESSMENTS: The primary safety outcome was time to death from any cause (non-inferiority for Respimat 2.5 and 5 µg vs HandiHaler 18 μg) and the primary efficacy outcome was time to first COPD exacerbation (superiority for Respimat 5 µg vs HandiHaler 18 μg). COPD exacerbations were defined as the worsening of two or more major respiratory symptoms (dyspnoea, cough, sputum, chest tightness or wheezing) with a duration of at least 3 days and requiring specified treatment changes. Mild exacerbations required a newly prescribed maintenance bronchodilator; moderate exacerbations required a prescription for antibiotics, systemic corticosteroids or both; and severe exacerbation required hospitalisation. Secondary outcomes included the number of COPD exacerbations, time to first moderate or severe exacerbation, time to first severe (hospitalised) exacerbation and time to non-fatal and fatal MACE (MACE included stroke, transient ischaemic attack, MI, sudden death, cardiac death, sudden cardiac death or fatal event in the system organ classes (SOCs) for cardiac and vascular disorders). SOCs were defined according to the Medical Dictionary for Regulatory Activities. Adverse event (AE) collection included all serious AEs, related AEs and AEs leading to discontinuation. All AEs and deaths were reviewed by an independent data and safety monitoring committee. In addition, all deaths were reviewed by an independent mortality adjudication committee (blinded to study-group assignments). For the non-fatal MACEs (stroke and MI) reported by site investigators, the accuracy of diagnosis/classification was verified by central reviewers, who were blinded to the study-group assignments. BODY.STATISTICAL ANALYSIS: HRs and 95% CIs for time-to-event end points were calculated using a Cox-proportional hazards regression model (with no covariate adjustment). Negative binomial regression models were used to compare annual exacerbation rates. Non-inferiority testing for the risk of death was performed using HandiHaler 18 μg as the reference treatment. Rate ratios and 95% CI were used to compare incidence rates. For analysis of death (including fatal MACE), events occurring during treatment and vital status follow-up (follow-up for deaths even if the patient discontinued early) were considered (vital status analysis), while for all other analyses (including MACE and COPD exacerbations), only events with onset in the on treatment period were noted. For the two primary outcomes, analyses on predefined subgroups specified by demographic variables, comorbid conditions, disease severity and concomitant medications, were performed. BODY.RESULTS: BODY.STUDY POPULATION: A total of 2784 patients from TIOSPIR who received stable tiotropium HandiHaler 18 µg prior to study entry were randomised and treated with blinded medication, and included in the analyses of mortality (tiotropium Respimat 2.5 µg: n=914; 5 µg: n=918; HandiHaler 18 µg: n=952). A total of 2782 patients were analysed for all other end points (two patients were excluded because they were from centres with data irregularities); baseline characteristics for this population are shown in table 1. Mean observation time (including vital status follow-up) was 858 days. In total, 679 patients (tiotropium Respimat 2.5 µg: n=229; 5 µg: n=211; HandiHaler 18 µg: n=239) prematurely discontinued from trial medication, including because of deaths. Table 1Patient baseline characteristics CharacteristicTiotropium Respimat2.5 μg (n=914)Tiotropium Respimat5 μg (n=917)Tiotropium HandiHaler18 μg (n=951)Gender, n (%)  Male 551 (60.3) 568 (61.9) 585 (61.5)  Female 363 (39.7) 349 (38.1) 366 (38.5) Age, mean years (SD) 67.3 (8.6) 67.4 (8.8) 66.9 (8.9) BMI, mean kg/m2 (SD) 27.2 (6.3) 26.9 (6.2) 26.8 (6.2) Current smoker, n (%) 262 (28.7) 278 (30.3) 290 (30.5) Smoking history, mean pack-years (SD) 51.9 (28.6) 51.4 (29.0) 52.7 (28.7) Postbronchodilator spirometry, mean (SD)  FEV1, L 1.21 (0.46) 1.25 (0.47) 1.24 (0.46)  FEV1, % predicted 45.8 (14.2) 46.7 (13.9) 46.1 (13.7)  FVC, L 2.62 (0.86) 2.65 (0.87) 2.64 (0.89)  Ratio of FEV1 to FVC 0.47 (0.12) 0.48 (0.11) 0.48 (0.11) GOLD stage, n (%)  I+II 367 (40.2) 398 (43.4) 375 (39.4)  III 411 (45.0) 398 (43.4) 443 (46.6)  IV 128 (14.0) 117 (12.8) 124 (13.0) Previous cardiac arrhythmia, n (%) 154 (16.8) 138 (15.1) 158 (16.6)  Atrial fibrillation or flutter 56 (6.1) 50 (5.5) 57 (6.0)  Bundle branch block 51 (5.6) 39 (4.3) 35 (3.7)  Ventricular fibrillation 3 (0.3) 3 (0.3) 4 (0.4)  Supraventricular tachycardia 7 (0.8) 7 (0.8) 16 (1.7)  Ventricular tachycardia 6 (0.7) 5 (0.5) 10 (1.1)  Bradycardia 14 (1.5) 18 (2.0) 25 (2.6)  Atrioventricular block 12 (1.3) 10 (1.1) 16 (1.7)  Other conduction disorders 25 (2.7) 27 (2.9) 28 (2.9) Previous MI, n (%) 79 (8.6) 91 (9.9) 99 (10.4) Previous stroke, n (%) 21 (2.3) 24 (2.6) 27 (2.8) Previous IHD or CAD, n (%) 154 (16.8) 149 (16.3) 184 (19.3) Taking CV medication, n (%) 554 (60.6) 551 (60.1) 568 (59.7)  β-blockers 169 (18.5) 178 (19.4) 190 (20.0)  Calcium channel blockers 164 (17.9) 175 (19.1) 174 (18.3)  Cardiac glycosides 22 (2.4) 19 (2.1) 28 (2.9)  ACE inhibitors 221 (24.2) 210 (22.9) 209 (22.0)  Angiotensin receptor blockers 111 (12.1) 128 (14.0) 127 (13.4)  Nitrates 40 (4.4) 46 (5.0) 49 (5.2)  Antiarrhythmics class I or III (sodium or potassium channel blockers) 6 (0.7) 8 (0.9) 15 (1.6)  Acetylsalicylic acid 276 (30.2) 300 (32.7) 281 (29.5)  Anticoagulants* 52 (5.7) 45 (4.9) 41 (4.3)  Antiplatelets 55 (6.0) 55 (6.0) 63 (6.6) Use of respiratory medication  LAMA 914 (100.0) 917 (100.0) 951 (100.0)  LABA 637 (69.7) 631 (68.8) 644 (67.7)  SABA 615 (67.3) 601 (65.5) 628 (66.0)  ICS 613 (67.1) 612 (66.7) 626 (65.8)  β-adrenergics 615 (67.3) 601 (65.5) 628 (66.0)  LRTA 50 (5.5) 49 (5.3) 56 (5.9)  Mucolytics 35 (3.8) 39 (4.3) 44 (4.6)  Supplemental oxygen 123 (13.5) 120 (13.1) 122 (12.8)  Xanthines 92 (10.1) 73 (8.0) 88 (9.3) Two patients from centres with data irregularities were excluded. *Includes vitamin K antagonists, direct thrombin inhibitors, factor Xa inhibitors. ACE, angiotensin converting enzyme; BMI, body mass index; CAD, coronary artery disease; CV, cardiovascular; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; GOLD, Global Initiative for Chronic Obstructive Lung Disease; ICS, inhaled corticosteroid; IHD, ischaemic heart disease; LABA, long-acting β22-agonist. Patient demographics and baseline characteristics were similar in the three tiotropium groups (table 1). The mean age of patients was 67.2 years, the majority (61.3%) were male, 29.8% were current smokers and mean postbronchodilator FEV1 was 46.2% of predicted. The majority of patients were Global Initiative for Chronic Obstructive Lung Disease Stages II (40.8%) and III (45.0%). Overall, 68.7% of patients were taking a long-acting β2-agonist at baseline and a similar proportion of patients (66.5%) were taking inhaled corticosteroids. The majority (60.1%) of patients were also receiving CV medications at baseline and 16.2% of patients had a history of cardiac arrhythmia (the most common type being atrial fibrillation or flutter; table 1). BODY.MORTALITY END POINTS: Both Respimat doses were non-inferior to HandiHaler with respect to the primary end point of time to death (vital status analysis)—Respimat 2.5 µg: HR 0.87; 95% CI 0.64 to 1.17; 5 µg: HR 0.79; 95% CI 0.58 to 1.07 (figure 1). In a sensitivity analysis, the risk of a fatal AE was lower in the Respimat 5 µg versus the HandiHaler group (HR 0.62; 95% CI 0.43 to 0.89; Respimat 2.5 µg: HR 0.78; 95% CI 0.55 to 1.09) (see E-figure 1). Causes of death were similar across the treatment groups, including death from cardiac disorders and MACE. Although the incidence of cardiac disorders was higher in the HandiHaler group (n=5 vs 2), the very small number of cardiac deaths, the small absolute risk difference and the wide CIs around the HRs, do not indicate any significant difference (table 2). Table 2Adjudicated primary causes of death (vital status analysis) VariableTiotropium Respimat2.5 μg (n=914)Tiotropium Respimat5 μg (n=918)Tiotropium HandiHaler18 μg (n=952)Rate ratio (95% CI)Tiotropium Respimat2.5 μg vs HandiHaler 18 μgTiotropium Respimat5 μg vs HandiHaler 18 μgAdjudicated causes of death, n (rate/100 patient-years) 77 (3.6) 71 (3.3) 92 (4.1) 0.87 (0.64 to 1.17) 0.79 (0.58 to 1.07)  Cardiac disorders 2 (0.1) 2 (0.1) 5 (0.2) 0.41 (0.08 to 2.14) 0.41 (0.08 to 2.10)  General disorders 12 (0.6) 14 (0.6) 18 (0.8) 0.69 (0.33 to 1.43) 0.79 (0.39 to 1.59)  Neoplasms benign, malignant and unspecified 25 (1.2) 16 (0.7) 18 (0.8) 1.44 (0.79 to 2.64) 0.91 (0.46 to 1.78)  Respiratory, thoracic and mediastinal disorders 29 (1.4) 26 (1.2) 33 (1.5) 0.91 (0.55 to 1.50) 0.80 (0.48 to 1.34)   COPD 28 (1.3) 25 (1.1) 28 (1.3) 1.04 (0.61 to 1.75) 0.91 (0.53 to 1.56)  Infections and infestations 6 (0.3) 6 (0.3) 4 (0.2) 1.55 (0.44 to 5.51) 1.53 (0.43 to 5.42)  Nervous system disorders 2 (0.1) 1 (0.0) 4 (0.2) 0.52 (0.09 to 2.83) 0.25 (0.03 to 2.28) Patients with fatal MACE, n (rate/100 patient-years) 11 (0.5) 13 (0.6) 20 (0.9) 0.57 (0.27 to 1.19) 0.66 (0.33 to 1.33)  Sudden death 5 (0.2) 8 (0.4) 5 (0.2) 1.04 (0.30 to 3.58) 1.63 (0.53 to 4.98)  Sudden cardiac death 2 (0.1) 0 (0.0) 5 (0.2) 0.41 (0.08 to 2.14) –  Cerebrovascular accident 1 (0.0) 1 (0.0) 4 (0.2) 0.26 (0.03 to 2.32) 0.25 (0.03 to 2.28)  Cardiac failure congestion 1 (0.0) 1 (0.0) 1 (0.0) 1.04 (0.06 to 16.56) 1.02 (0.06 to 16.29)  Acute myocardial infarction 1 (0.0) 0 (0.0) 0 (0.0) – –  Aortic dissection 0 (0.0) 0 (0.0) 1 (0.0) – –  Aortic valve stenosis 0 (0.0) 0 (0.0) 1 (0.0) – –  Arteriosclerosis 0 (0.0) 1 (0.0) 0 (0.0) – –  Cardiac failure chronic 0 (0.0) 0 (0.0) 1 (0.0) – –  Cardiac valve disease 0 (0.0) 1 (0.0) 0 (0.0) – –  Cor pulmonale 0 (0.0) 0 (0.0) 1 (0.0) – –  Myocardial infarction 0 (0.0) 0 (0.0) 1 (0.0) – –  Peripheral vascular disorder 0 (0.0) 1 (0.0) 0 (0.0) – –  Subarachnoid haemorrhage 1 (0.0) 0 (0.0) 0 (0.0) – – Time at risk adjusted rate ratios of adjudicated causes of death by treatment, MedDRA (V.16.0) system organ class and preferred term. COPD, chronic obstructive pulmonary disease; MACE, major adverse cardiovascular event (stroke, transient ischaemic attack, myocardial infarction, sudden death, cardiac death, sudden cardiac death or fatal event in the system organ classes for cardiac and vascular disorders); MedDRA, Medical Dictionary for Regulatory Activities. Figure 1Kaplan–Meier plot of time to death by treatment (vital status analysis). No significant treatment interactions were observed for either Respimat dose compared with HandiHaler in the subgroups of patients with baseline cardiac arrhythmia (Respimat 2.5 µg: HR 1.03; 95% CI 0.58 to 1.84; 5 µg: HR 0.83; 95% CI 0.44 to 1.56) or cardiac history defined as history of MI, ischaemic heart disease/coronary artery disease, cardiac arrhythmia or heart failure (Respimat 2.5 µg: HR 1.01; 95% CI 0.65 to 1.57; 5 µg: HR 0.93; 95% CI 0.59 to 1.46) (see E-figure 2a, b). BODY.EXACERBATION EFFICACY: There was no difference in the primary end point of time to first COPD exacerbation (on treatment) in either of the Respimat groups versus HandiHaler (HR 1.03; 95% CI 0.92 to 1.16; and HR 0.96; 95% CI 0.86 to 1.08 for Respimat 2.5 and 5 µg vs HandiHaler, respectively) (table 3 and E-figure 3a). The median time to first exacerbation was 390 and 466 days in the Respimat 2.5 and 5 µg groups, respectively, and 418 days in the HandiHaler group. The number of exacerbations was similar between the treatment groups (adjusted rate/patient-year: 0.83, 0.83 and 0.81 in the Respimat 2.5 or 5 µg and HandiHaler groups, respectively). Table 3Risk and rate of exacerbations (on treatment analysis*) VariableTiotropium Respimat2.5 μg (n=914)Tiotropium Respimat5 μg (n=917)Tiotropium HandiHaler18 μg (n=951)HR (95% CI); p valueTiotropium Respimat2.5 μg vs HandiHaler 18 μgTiotropium Respimat5 μg vs HandiHaler 18 μgAny exacerbation  Patients with event, n (%) 573 (62.7) 560 (61.1) 578 (60.8) 1.03 (0.92 to 1.16); p=0.614 0.96 (0.86 to 1.08); p=0.517  Number of events 1484 1508 1548  Adjusted rate of events/patient-year (95% CI) 0.83 (0.77 to 0.91) 0.83 (0.76 to 0.90) 0.81 (0.74 to 0.87) Moderate-to-severe exacerbation  Patients with event, n (%) 561 (61.4) 550 (60.0) 571 (60.0) 1.01 (0.90 to 1.14); p=0.817 0.96 (0.85 to 1.07); p=0.441  Number of events 1462 1474 1525  Adjusted rate of events/patient-year (95% CI) 0.82 (0.75 to 0.89) 0.81 (0.74 to 0.88) 0.79 (0.73 to 0.86) Severe (hospitalised) exacerbation  Patients with event, n (%) 172 (18.8) 173 (18.9) 172 (18.1) 1.04 (0.85 to 1.29); p=0.690 1.03 (0.84 to 1.28); p=0.760  Number of events 264 283 267  Adjusted rate of events/patient-year (95% CI) 0.15 (0.13 to 0.18) 0.16 (0.13 to 0.19) 0.14 (0.12 to 0.17) Two patients from centres with data irregularities were excluded. *Includes first day after treatment stop. No difference was observed between the Respimat-treated population and HandiHaler groups with respect to the secondary end points of time to first moderate-to-severe exacerbation and time to first severe (hospitalised) exacerbation (table 3; E-figure 3b, c). The frequency of moderate-to-severe and hospitalised exacerbations was low and similar between treatment groups (table 3). No significant treatment interactions were observed for either of the Respimat doses versus HandiHaler for the predefined subgroups with respect to exacerbation risk. BODY.OTHER SAFETY END POINTS: Serious AEs were reported in 42.5% of patients, with similar frequencies between each treatment group (table 4). Table 4Summary of AEs and MACE by treatment (on treatment analysis*) VariableTiotropium Respimat2.5 μg (n=914)Tiotropium Respimat5 μg (n=917)Tiotropium HandiHaler18 μg (n=951)Any AE, n (%) 711 (77.8) 721 (78.6) 737 (77.5) Drug-related AEs, n (%) 73 (8.0) 66 (7.2) 75 (7.9)  Respiratory, thoracic and mediastinal disorders 42 (4.6) 37 (4.0) 31 (3.3)  Gastrointestinal disorders 16 (1.8) 15 (1.6) 18 (1.9)  Nervous system disorders 11 (1.2) 3 (0.3) 6 (0.6)  Cardiac disorders 2 (0.2) 1 (0.1) 8 (0.8) Serious AEs, n (%)† 373 (40.8) 399 (43.5) 409 (43.0)  Respiratory, thoracic and mediastinal disorders 198 (21.7) 203 (22.1) 212 (22.3)  Infections and infestations 118 (12.9) 115 (12.5) 110 (11.6)  Neoplasms—benign, malignant and unspecified (including cysts and polyps) 71 (7.8) 73 (8.0) 65 (6.8)  Cardiac disorders 50 (5.5) 56 (6.1) 71 (7.5)  Nervous system disorders 26 (2.8) 31 (3.4) 34 (3.6)  General disorders and administration-site conditions 18 (2.0) 24 (2.6) 30 (3.2)  Renal and urinary disorders 11 (1.2) 12 (1.3) 15 (1.6) Patients with MACE, n (%)‡ 34 (3.7) 31 (3.4) 57 (6.0)  Myocardial infarction 8 (0.9) 6 (0.7) 11 (1.2)  Cerebrovascular accident 6 (0.7) 6 (0.7) 10 (1.1)  Transient ischaemic attack 4 (0.4) 11 (1.2) 6 (0.6) Patients with MACE, n (%)§ 32 (3.5) 31 (3.4) 46 (4.8) Two patients from centres with data irregularities were excluded. *Includes thirty days after treatment stop. †Frequency of patients with serious AEs, as determined by the investigator, occurring in 15 or more patients at the MedDRA (V.16.0) preferred term level by treatment and primary system organ class. ‡Frequency of patients with AEs classified as MACE, as determined by the investigator, occurring in 10 or more patients at the preferred term level by treatment and primary system organ class. §Frequency of patients with AEs classified as MACE based on adjudicated causes of death. AE, adverse event; MACE, major adverse cardiovascular event (stroke, transient ischaemic attack, myocardial infarction, sudden death, cardiac death, sudden cardiac death or fatal event in the system organ classes for cardiac and vascular disorders); MedDRA, Medical Dictionary for Regulatory Activities. For the secondary safety end points of time to first MACE, there was no significant difference between the Respimat and HandiHaler groups (table 4 and E-figure 4a, b). The incidence of first MACE was 3.5% in the Respimat 2.5 µg group, 3.4% in the 5 µg group and 4.8% in the HandiHaler group (on treatment analysis, based on adjudicated causes of death) (table 4) (HR 0.73; 95% CI 0.47 to 1.15, p=0.179; and HR 0.69; 95% CI 0.44 to 1.08, p=0.107 for Respimat 2.5 and 5 µg vs HandiHaler, respectively). BODY.DISCUSSION: This post hoc analysis of TIOSPIR demonstrated that patients receiving stable (≥2 months) treatment with tiotropium HandiHaler 18 µg at baseline who changed to treatment with tiotropium Respimat 2.5 or 5 µg were at a similar (though numerically lower) risk of death and MACE as patients who continued to receive HandiHaler 18 µg. It also found that recipients of tiotropium Respimat had a similar risk of exacerbation as those receiving tiotropium HandiHaler. These findings are consistent with the TIOSPIR study results. Evidence from previous studies, primarily retrospective analyses, has raised concerns that tiotropium delivered by Respimat may be associated with adverse CV effects in patients with CV comorbidities.9–14 17 Approximately 16% of patients in this post hoc analysis had a history of cardiac arrhythmias. The subgroup analyses in patients with a history of cardiac arrhythmia and those with cardiac history, predefined in the main study, demonstrated no significant difference between Respimat and HandiHaler recipients with respect to mortality and exacerbation risk; in line with the main study, no increased risk could be identified, even in the subgroup of patients who changed from HandiHaler to Respimat. There was a 38% reduced risk of fatal AEs in the Respimat 5 µg group versus the HandiHaler group. Together with a numerical (though not statistically significant) imbalance in favour of both Respimat doses with respect to incidence of first MACE, this argues against any deleterious effect of tiotropium when given using Respimat. Importantly, previous studies of long-term treatment of patients with COPD with tiotropium Handihaler in the 4-year UPLIFT study did not indicate any adverse effect on MACE or fatal MACE versus placebo.8 Other recent studies have provided additional support for the safety of Respimat in patients with COPD. A pharmacokinetic study4 showed that, although the overall profile was similar, systemic exposure to tiotropium following the use of tiotropium Respimat was slightly lower compared with tiotropium HandiHaler. A post hoc analysis of mortality and exacerbation data from six clinical trials of tiotropium Respimat and tiotropium HandiHaler (including UPLIFT and TIOSPIR) showed similar effects on both, mortality and exacerbations, between Respimat 5 µg and HandiHaler 18 µg.21 In addition, risk of exacerbation was lower for both tiotropium formulations versus placebo, with numerically higher efficacy for Respimat 5 μg.21 In another study, combined analysis of all trials from the tiotropium clinical trial database involving Holter-ECG monitoring in patients with COPD did not demonstrate any clinically relevant differences between Respimat and HandiHaler with respect to changes in heart rate or in the proportion of patients experiencing supraventricular or ventricular premature beats while on tiotropium.22 Finally, a pooled analysis of AE data from 28 randomised trials of HandiHaler and seven Respimat studies did not indicate a significantly increased risk of fatal AEs or fatal/non-fatal MACE with either inhaler versus placebo.23 A recent drug utilisation study investigated the influence of factors on the probability of receiving tiotropium HandiHaler or Respimat and determined the existence of a channelling effect both in incident users and in patients switching from the HandiHaler to the Respimat formulation.20 It found that comorbidities and disease severity influence the likelihood of switching from HandiHaler to Respimat, and called for further studies comparing the safety profile of the two formulations.20 The present study addresses any safety and efficacy concerns regarding the switch between formulations. TIOSPIR is the largest COPD study conducted to date, comprising over 34 000 patient-years of exposure to tiotropium.19 Clinical trials are often criticised for not recruiting patients typical of the population treated in routine clinical practice. The liberal inclusion criteria of the TIOSPIR study permitted a large number of patients to be included, with a wide range of disease severities and history of cardiac disorders; it was thus representative of a typical COPD population (although it excluded patients with severe, unstable CV disease or moderate-to-severe renal impairment). In addition, the large size of the population allowed for statistically powerful subgroup analyses. The results of TIOSPIR can be regarded as more robust than findings from meta-analyses or database studies, which typically evaluate less frequent outcomes from clinical studies that may not have been collected as rigorously as primary or secondary outcome measures.24 Database studies evaluating rare events may not fully adjust confounding by severity; for example, it is difficult to interpret the independent effect of tiotropium Respimat without a detailed assessment of underlying COPD and CV risk.24 Furthermore, differences often exist in the populations studied, doses of treatment and length of follow-up. A limitation of the study is the post hoc design involving a subgroup of patients with a lower number of patients than in the main study. The design of this post hoc analysis does not allow for an evaluation of treatment initiation with the two inhalers in anticholinergic-naïve patients; results for these patients will be reported elsewhere. This post hoc analysis was restricted to patients who were receiving tiotropium HandiHaler 18 µg treatment for at least 2 months prior to randomisation. It is unfortunate that data on the duration of treatment outside of the 2-month cut-off period were not collected, as this would facilitate better characterisation of the patients who were included in the study. For example, patients who had been treated with tiotropium HandiHaler for longer than 2 months could be considered more ‘stable’ on this treatment than patients who were recently initiated on tiotropium. While the current analysis excluded patients with moderate-to-severe renal impairment, placebo-controlled studies of patients with moderate renal impairment do not indicate an increased risk in these patients with the use of tiotropium Respimat.25 26 A pooled safety analysis of 22 Phase III and IV clinical trials of tiotropium HandiHaler and Respimat (N=10 805 patients) demonstrated no trend for increased incidence rate ratios of AEs with worsening renal function for either formulation.25 26 In this current post hoc analysis, the group of patients with severe renal impairment was too small to be analysed. BODY.CONCLUSION: This post hoc analysis of the pivotal TIOSPIR trial provides additional evidence for the safety of tiotropium delivered by Respimat, which demonstrated a similar safety profile to tiotropium HandiHaler in patients with COPD, including those with a history of cardiac disorders. Based on this analysis, it can be concluded that it is safe to switch patients from tiotropium HandiHaler to tiotropium Respimat, and that the efficacy is maintained over the switch.

TITLE: Effect of Insulin Glargine and n-3FA on Carotid Intima-Media Thickness in People With Dysglycemia at High Risk for Cardiovascular EventsThe Glucose Reduction and Atherosclerosis Continuing Evaluation Study (ORIGIN-GRACE) ABSTRACT.OBJECTIVE: To evaluate the effects of insulin glargine and n-3 polyunsaturated fatty acid (n-3FA) supplements on carotid intima-media thickness (CIMT). ABSTRACT.RESEARCH DESIGN AND METHODS: We enrolled 1,184 people with cardiovascular (CV) disease and/or CV risk factors plus impaired fasting glucose, impaired glucose tolerance, or early type 2 diabetes in a randomized multicenter 2 × 2 factorial design trial. Participants received open-label insulin glargine (targeting fasting glucose levels ≤5.3 mmol/L [95 mg/dL]) or standard glycemic care and double-blind therapy with a 1-g capsule of n-3FA or placebo. The primary trial outcome was the annualized rate of change in maximum CIMT for the common carotid, bifurcation, and internal carotid artery segments. Secondary outcomes were the annualized rates of change in maximum CIMT for the common carotid and the common carotid plus bifurcation, respectively. Baseline followed by annual ultrasounds were obtained during a median follow-up of 4.9 years. ABSTRACT.RESULTS: Compared with standard care, insulin glargine reduced the primary CIMT outcome, but the difference was not statistically significant (difference = 0.0030 ± 0.0021 mm/year; P = 0.145) and significantly reduced the secondary CIMT outcomes (differences of 0.0033 ± 0.0017 mm/year [P = 0.049] and 0.0045 ± 0.0021 mm/year [P = 0.032], respectively). There were no differences in the primary and secondary outcomes between the n-3FA supplement and placebo groups. ABSTRACT.CONCLUSIONS: In people with CV disease and/or CV risk factors and dysglycemia, insulin glargine used to target normoglycemia modestly reduced CIMT progression, whereas daily supplementation with n-3FA had no effect on CIMT progression. Atherosclerosis is the major cause of death and disability in people with type 2 diabetes and lesser degrees of dysglycemia (1,2). Large epidemiological studies show consistent independent associations between glycemia and cardiovascular (CV) risk (1–4), and the metabolic abnormalities associated with dysglycemia promote atherosclerosis (5). Exogenous insulin can provide effective glycemic control, but its effects on atherosclerosis are unknown. Moreover, some studies suggest possible proatherogenic effects (6,7). Essential long-chain n-3 polyunsaturated fatty acids (n-3FA) may have beneficial effects on atherosclerosis (8). Higher intake of fish or n-3FA supplements is associated with lower rates of coronary heart disease and death (9,10) and lower atherosclerotic burden (11,12), and some, but not all, previous trials reported reduced CV events in patients receiving n-3FA supplements (13–16). The effects of these supplements on human atherosclerosis progression were evaluated in a few small studies, which were inconclusive (17–21). Therefore, we evaluated the effects of insulin glargine and n-3FA supplements on carotid intima-media thickness (CIMT) in people with dysglycemia and additional risk factors for atherosclerosis progression in a substudy of the Outcome Reduction with an Initial Glargine Intervention (ORIGIN) trial (22–24). BODY.RESEARCH DESIGN AND METHODS: BODY.STUDY DESIGN AND STUDY POPULATION: The Glucose Reduction and Atherosclerosis Continuing Evaluation substudy of ORIGIN (ORIGIN-GRACE) is an investigator-initiated, randomized, controlled, parallel-group study with a 2 × 2 factorial design. Clinical eligibility criteria, study interventions, and follow-up procedures are those described in detail previously (22–24), with the addition of serial carotid ultrasound (CUS) examinations. The study was conducted at 32 ORIGIN centers in seven countries, selected based on interest and availability of adequate ultrasound equipment, which met preset technical specifications, and expert sonographers, who met predefined performance criteria. Funding and regulatory support were provided by Sanofi, and capsules containing n-3FA and placebo were provided by Pronova BioPharma, Norway. Project coordination, data management, and statistical analyses were independently provided by the Population Health Research Institute in Hamilton, Canada, which was also the site for the Core CUS Laboratory. The study was approved by the ethics review boards of all participating institutions, and all participants provided written informed consent. Between 5 February 2004 and 27 December 2005, we enrolled people ≥50 years of age with dysglycemia, defined as early diabetes on no more than one oral glucose-lowering drug, impaired glucose tolerance (IGT), or impaired fasting glucose (IFG) and with known CV disease and/or CV risk factors (detailed clinical eligibility criteria are published [22–24] and are summarized in Supplementary Appendix 2). In addition, patients were required to have an adequate baseline CUS examination, defined as a scan allowing reliable measurements from a minimum of four predefined carotid arterial segments, as per the Core Ultrasound Laboratory’s review. BODY.RANDOMIZATION, STUDY INTERVENTIONS, ALLOCATION CONCEALMENT, AND FOLLOW-UP: Eligible participants were randomized by an automated telephone randomization system (using randomly varying block sizes, stratified by center) according to a 2 × 2 factorial design to 1) either insulin glargine (Lantus; Sanofi) or standard approaches to glycemic control and 2) either n-3FA (Omacor 1 g; Pronova BioPharma AS, Lysaker, Norway) containing eicosapentaenoic acid (EPA) 465 mg and docosahexaenoic acid (DHA) 375 mg or matching placebo containing ∼1 g olive oil. The randomization sequence was concealed, and all study personnel (except one unblinded statistician at the project office) were unaware of the randomization procedure. The insulin glargine arm of the study used a prospective, randomized, open, blinded end point design (PROBE), so that study participants and site investigators were not blinded but all personnel at the Core CUS Laboratory and all other study personnel and investigators involved in event adjudication and data analysis were blinded to treatment assignment. Participants assigned to insulin glargine added one evening injection to their glycemic-control regimen and increased the dose at least once weekly, targeting a self-measured fasting plasma glucose (FPG) level of 5.3 mmol/L (95 mg/dL) or less. Participants assigned to standard care were treated on the basis of the investigator’s best judgment and local guidelines. The n-3FA arm of the trial was blinded to study participants, site investigators, and all local and central trial personnel. Study visits occurred at 0.5, 1, 2, and 4 months after randomization and every 4 months thereafter. FPG and glycated hemoglobin (HbA1c) levels were measured at 4 months, 8 months, and annually, and fasting lipid levels were measured at baseline, 2 years, and study end in all participants. A food frequency questionnaire was administered at randomization, at 2 years, and at the end of the study, and the dietary intake of EPA and DHA was calculated using the Department of Agriculture National Nutrient Database for Standard Reference, release 23 (USDA Food Search for Windows, version 1.0). BODY.QUANTITATIVE CAROTID ULTRASONOGRAPHY: CUS examinations were performed at baseline and yearly thereafter until 1–1.3 years prior to the final ORIGIN study visit (average six scans per participant). The ultrasound methods have been reviewed in detail previously (25). Sonographer training, quality control, and CIMT measurements (readings) were performed by the Core Laboratory. Standardized and validated scanning and measurement protocols were used. All CUS scans were performed by trained and certified sonographers using high-resolution imaging systems with linear array transducers operating at a fundamental frequency of at least 7.5 MHz (for each subject, the same ultrasound imaging system and transducer were used throughout the study). A transverse B-mode scan was followed by a circumferential longitudinal scan, aimed at recording the maximum CIMT in each of 12 carotid artery segments (1-cm long), which were defined relative to the carotid flow divider as the near and far walls of the internal, bifurcation, and common left and right carotid arteries. Three trained and certified readers unaware of treatment assignment performed all measurements using the Image-ProPlus software (Media Cybernetics, Silver Spring, MD). For each carotid arterial segment, the reader selected a minimum of three frames showing the thickest CIMT. The leading edge (far wall) and the trailing edge (near wall) of the boundaries between the lumen and media and the media and adventitia were traced, obtaining measurements of segment maximum and mean CIMT. Scans were read in batch fashion and in random order for each individual in order to exclude potential reader drift in measurements and to ensure use of similar anatomical landmarks. Batches were read by a single reader to avoid interreader variability. Intraclass correlation coefficients for 250 paired baseline CUS examinations performed maximum 10 days apart were 0.98 for the average maximum CIMT from 12 carotid artery segments and ranged from 0.93 to 0.98 for segment maximum and mean CIMT measurements. At study end, intraclass correlation coefficients evaluated on 26 paired CUS examinations were 0.95 for the average maximum CIMT from 12 carotid artery segments and ranged from 0.87 to 0.98 for segment maximum and mean CIMT measurements. Completeness of data by carotid arterial segment was as follows: 99% for the common carotid far wall, 96% for the common carotid near wall, 94% for the bifurcation far wall, 91% for the bifurcation near wall, 71% for the internal far wall, and 55% for the internal near wall. BODY.STUDY OUTCOMES: The primary outcome was the annualized change in the maximum CIMT for the near and far walls of the right and left common carotid, bifurcation, and internal carotid artery segments (12 carotid artery segments) based on all scans performed during the study. The secondary outcomes were the annualized change in the maximum common carotid CIMT (four segments, the near and far walls of the right and left common carotid artery segments) and the annualized change in the maximum CIMT for the common carotid and bifurcation (eight segments, the near and far walls of the right and left common carotid and bifurcation). An additional CUS outcome was the annualized change in the maximum far wall CIMT (six segments, the right and left far walls of the common carotid, bifurcation, and internal carotid artery segments). CV outcome events were collected and adjudicated as part of the parent ORIGIN trial. BODY.STATISTICAL ANALYSIS: Sample size calculations showed that 800 participants would provide 80% power to detect a 25% treatment effect at the margins of the factorial, based on a repeated-measures analysis, assuming a control progression rate of 0.017 mm/year for the primary outcome, five CUS measurements per study participant, baseline average maximum CIMT of 1.15 in the treatment and control groups, a correlation between repeated measurements of 0.84 (between variance = 0.09613 and total variance = 0.11487, as estimated from another CIMT trial performed by our group in a high-risk population, the Study to Evaluate Carotid Ultrasound with Ramipril and vitamin E [SECURE]) (26), and no significant interaction between the treatments. In light of possible lower CIMT progression rates, and allowing for a 5% attrition rate, we increased the sample size, which was set a priori to 1,100 participants. All analyses are by intention to treat and were performed in SAS version 9.1 for Solaris. The primary analyses compared the primary, secondary, and additional CUS outcomes between the insulin glargine versus standard glycemic care and between the n-3FA versus the placebo groups, after confirming that there was no significant interaction between the study treatments for the primary, secondary, or additional CUS outcomes (P = 0.496, 0.749, 0.789, and 0.353, respectively, for interaction terms in the regression models). The main efficacy analysis included all participants with at least one adequate CUS examination after the baseline scan. As previously described (25,27–29), a repeated-measures linear mixed-effects model was used to analyze the annualized rate of change in maximum CIMT, including all segment maximum measurements for each patient as the dependent variables, with random intercepts and slopes as a function of time and fixed effects for geographic region, age, sex, treatment assignment for the other arm of the factorial design, carotid segment, treatment, time, and interaction between time and treatment. Testing was two sided and conducted with a 5% type I error rate. Similar analyses were used for the secondary and additional CIMT outcomes. Additional models were computed with the addition of fixed effects for baseline and average on treatment HbA1c, FPG, triglyceride, LDL cholesterol, HDL cholesterol, and blood pressure (BP) levels (entered individually and sequentially). Prespecified subgroup analyses were performed for age (above and below 65 years), sex, baseline glycemic status (diabetes or no diabetes), CV history (previous CV event or no previous CV event), baseline CIMT, HbA1c, FPG, and triglyceride levels (above and below median), and baseline treatment with statins and ACE inhibitors or angiotensin receptor blockers (ARBs). Laboratory measurements were analyzed by ANCOVA, using terms for treatment assignment to the other arm of the factorial, baseline metabolic status (known diabetes, new diabetes, or IFG/IGT), status with respect to a history of CV disease, and the baseline laboratory measurement as covariate. BP and heart rate changes were analyzed by repeated-measures analyses, and clinical outcomes were analyzed as part of the main ORIGIN trial, as previously described (22–24). BODY.RESULTS: BODY.STUDY POPULATION, ADHERENCE, AND SAFETY: A total of 1,184 participants at 32 centers met clinical and CUS eligibility criteria; 580 were randomly assigned to insulin glargine, 604 to standard glycemic care, 585 to n-3FA, and 599 to placebo. Of these, 25 died before the scheduled first postrandomization CUS at the 1 year visit and 68 did not have any adequate follow-up CUS examination. In total, 1,091 patients (92.2%) had at least one adequate follow-up CUS examination and are evaluated in the primary efficacy analysis (533 allocated to insulin glargine and 558 to standard glycemic care; 539 to n-3FA and 552 to placebo). All 1,184 participants were followed for safety and clinical outcomes for a median of 6.2 years (interquartile range [IQR] 5.8–6.5). The median time from the baseline to the study end CUS was 4.9 years (IQR 3.0–5.0). At study end, vital status was unknown in two participants (Supplementary Fig. 1). Baseline characteristics of the 1,184 participants randomized in the GRACE study were well balanced between the treatment groups, were generally similar to those of the entire ORIGIN study population (except for the geographic distribution, with proportionally more participants from South America and fewer from Europe in GRACE compared with ORIGIN), and confirm participants’ high risk. Baseline CIMT did not differ significantly between the treatment groups (Table 1). Table 1Baseline characteristics by treatment group Adherence to insulin glargine at 1, 2, 3, 4, and 5 years and at study end was 94.0, 93.0, 91.0, 90.1, 89.3, and 86.3%, respectively. Nonstudy insulin was used at study end in 3.1% of patients in the insulin glargine and 10.4% in the standard care group. For the n-3FA supplement arm of the trial, adherence rates were 97.2% for active n-3FA and 97.3% for placebo at 1 year, 96.6 and 95.8% at 2 years, 95.5 and 94.6% at 3 years, 94.5 and 94.5% at 4 years, 93.8 and 94.3% at 5 years, and 91.4 and 92.6%, respectively, at study end. A total of 91 participants (15.7%) permanently discontinued insulin glargine, most frequently due to patient preference (76 patients) and hypoglycemia (9 patients). Sixty-six (11.3%) participants in the n-3FA group and 64 (10.7%) in the placebo group permanently discontinued the study drug, most frequently due to patient preference (45 and 43 patients, respectively), abdominal discomfort (4 and 2 patients, respectively), and lower gastrointestinal problems (2 and 4 patients, respectively). Intracranial bleeding occurred in four patients receiving n-3FA and four patients in the placebo group. Baseline characteristics, adherence rates, and side effects of the 1,091 patients included in the primary efficacy analysis were similar to those of the entire GRACE study population (Supplementary Tables 1 and 2). BODY.CHANGES IN CV RISK FACTOR LEVELS: Compared with the standard care group, FPG, HbA1c, and triglyceride levels were lower in the insulin glargine group at 2 years and at study end (Fig. 1). There were no significant differences in BP, heart rate, and in total, LDL, and HDL cholesterol levels (Supplementary Fig. 2). There were no significant differences in BP, heart rate, lipid, and glycemia measures between the n-3FA and the placebo groups (Supplementary Fig. 3). Dietary n-3FA consumption remained similar in the n-3FA and placebo groups at 2 years (median 58.8 mg/day [IQR 0.7–230] and 60.3 mg/day [0.3–230.2]) and at study end (median 95.3 mg/day [0.7–287.1] and 93.1 mg/day [1.5–268.4]). Figure 1Changes in levels of FPG (A), HbA1c (B), and triglycerides (C) in patients receiving insulin glargine and standard care. BODY.PRIMARY EFFICACY ANALYSIS: TREATMENT EFFECTS ON CIMT: For the insulin glargine arm of the study, we observed a statistically nonsignificant reduction in CIMT progression for the primary outcome and significant differences, favoring insulin glargine therapy for the secondary CIMT outcomes. These findings did not differ significantly in models adjusting for baseline and average on treatment FPG, HbA1c, lipids, and BP. There were no significant differences for the primary, secondary, and additional CIMT outcomes between the n-3FA and placebo groups (Table 2 and Fig. 2). The effect of both interventions on the primary outcome was similar across predefined subgroups (Supplementary Fig. 4) and across geographic regions. Table 2Main efficacy analysis: annualized changes (slopes) for the primary, secondary, and additional efficacy outcomes* Figure 2Changes in the primary and secondary CIMT outcomes by treatment group for the insulin glargine (A) and n-3FA (B) arms of the trial (slopes of carotid intima-media change and 95% CIs). BIF, bifurcation; CC, common carotid. BODY.CV EVENTS: Major CV events (defined as CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalized heart failure) occurred in 29.5% of participants in the insulin glargine and 29.0% in the standard care groups and in 28.4% in the n-3FA and 29.9% in the placebo groups, respectively. There were also no differences in all-cause and CV death, myocardial infarction, stroke, revascularization, and angina rates. More robust data on the effect of the study drugs on clinical events are provided by the larger, parent ORIGIN trial (23,24). BODY.CONCLUSIONS: ORIGIN-GRACE is the largest reported clinical trial that evaluated the effects of insulin and n-3FA supplements on atherosclerosis progression. Insulin glargine, a basal insulin, titrated to achieve normoglycemia, significantly lowered FPG, HbA1c, and triglyceride levels and had consistent favorable effects on CIMT progression, whereas n-3FA supplements had no significant effect on glycemia, lipids, and CIMT. Once-daily subcutaneous injections with insulin glargine were acceptable to patients, as evidenced by the high adherence rates, were generally safe, and resulted in excellent glycemic control, with mean FPG and HbA1c levels of 5.2 mmol/L and 6.0% at 1 year and 5.3 mmol/L and 6.5% at 5 years, respectively. They modestly lowered triglyceride levels and had no significant effects on BP, heart rate, and cholesterol levels. There was a statistically nonsignificant 11% reduction in the slope of CIMT progression for the primary outcome and significant 20 and 18%, respectively, for the secondary outcomes, with similar trends, 15% reduction, for the additional CIMT end point. These differences in CIMT progression could not be explained by differences in FPG and HbA1c, suggesting that these may be independent of glucose lowering. We selected a priori as our primary study outcome the annualized change in the maximum CIMT from all 12 carotid arterial segments and found a statistically nonsignificant trend toward a lower progression rate with insulin glargine. However, the optimal CIMT outcome in clinical trials remains controversial (30,31). Whereas some groups favor the change in the maximum CIMT across 12 carotid segments (as a more comprehensive approach), others favor changes in our predefined secondary outcomes (i.e., the common carotid and the common carotid plus bifurcation segments), due to fewer missing data and higher reproducibility (especially for the common carotid artery), or our additional CIMT outcome, because of higher accuracy for far wall measurements (30,31). Indeed, in our trial, missing data were highest for the internal carotid artery segments. Our findings provide further data supporting the CV safety of insulin glargine. The parent ORIGIN trial found no increase in clinical CV events with insulin glargine after 6.2 years, and the GRACE substudy found no adverse effects on atherosclerosis after 5 years. Diabetic patients frequently require glycemic control for decades. The lack of adverse effects of basal insulin on the arterial wall over 5 years suggests that longer-term therapy is likely to remain safe with regards to CV outcomes and may result in clinical CV benefits. The U.S. Food and Drug Administration recently mandated proof of CV safety as a major requirement for32). The findings of ORIGIN and its atherosclerosis substudy, GRACE, provide a very robust body of evidence for the CV safety of insulin glargine. In experimental studies, insulin reduced inflammatory markers (5,33) and improved endothelial function (34) and atherogenic plasma lipid patterns (35), although some studies suggested a possible proatherogenic effect of exogenous insulin in insulin-resistant states associated with compensatory hyperinsulinemia, possibly by stimulating cell proliferation through the MAPK pathway (6,7). There are surprisingly few studies on the effects of insulin on human atherosclerosis progression. The long-term follow-up of the Diabetes Control and Complications Trial (DCCT) cohort, the Epidemiology of Diabetes Interventions and Complications (EDIC) study, which compared CIMT in 1,229 patients with type 1 diabetes, 611 who had been randomly assigned to conventional diabetes treatment during the DCCT and 618 to intensive insulin treatment, found that 6 years after completion of the randomized DCCT intervention trial, CIMT was significantly lower in the group that had received intensive insulin therapy during the trial (36). Several studies confirmed the presence of increased CIMT in people with type 2 diabetes or prediabetes (37). However, there are only a few small, and therefore not reliable, intervention trials with insulin on atherosclerosis progression (38,39). Clinical CV end point trials conducted prior to ORIGIN also fail to provide clear answers regarding the impact of exogenous insulin on CV events. Extended follow-up of the UK Prospective Diabetes Study (UKPDS) trial found a legacy effect, with 15% reduction in myocardial infarction and 13% reduction in death among people with new-onset type 2 diabetes treated with insulin and sulfonylurea (40). Subsequent large-outcome trials of more versus less intense glucose lowering failed to demonstrate clear CV benefits, although insulin was used in both study groups in these strategy trials (41). The ORIGIN trial found a neutral effect of insulin glargine on CV events over 6.2 years (23). The GRACE substudy shows a modest decrease in carotid atherosclerosis consistent with EDIC, providing a rationale for an extended follow-up to assess whether the observed differences in atherosclerosis persist and whether these differences translate into clinical event reduction. n-3FA were reported to have several potentially antiatherogenic effects, such as improving endothelial function, lowering BP, inhibiting platelet aggregation, reducing triglycerides, and raising HDL2 cholesterol levels (8). Observational studies indicate associations between n-3FA intake and lower risk of CV events, and some clinical trials found clinical CV event reduction with n-3FA supplements (8–15). We found no significant effects of daily intake over 4.9 years of 1 g of n-3FA supplements on BP, lipid levels, and CIMT, and the parent ORIGIN trial found no effect on clinical CV events over 6.2 years (24). This is consistent with the results of previous smaller studies examining the effects on carotid and coronary atherosclerosis (17,20,21), as well as a recent meta-analysis of the effects of n-3FA on clinical outcomes (16). It is unclear if these findings are unique to our study population and the n-3FA dose and formulation used. Ongoing clinical end point trials will provide further insight into the role of n-3FA supplements in CV prevention (24). Moreover, our study does not address the CV effects of dietary fish consumption. In conclusion, treatment with basal insulin glargine over 4.9 years had a modest beneficial effect, whereas 1 g of n-3FA supplements had no impact on carotid atherosclerosis. Our findings confirm the CV safety of insulin and raise the possibility that longer-term treatment might result in CV event reduction. This hypothesis is currently under evaluation in the ORIGIN passive extended follow-up, the ORIGIN and Legacy Effects (ORIGINALE) study. Our findings do not support the use of n-3FA supplements in high-risk people with dysglycemia.

TITLE: Effect of dexmedetomidine infusion for intravenous patient-controlled analgesia on the quality of recovery after laparotomy surgery ABSTRACT.BACKGROUND: The Quality of Recovery-15 (QoR-15) is a patient-centered questionnaire to evaluate the recovery after surgery and anesthesia. Dexmedetomidine has sedative, analgesic, antiinflammatory and inhibitory sympathetic effects, which may contribute to early recovery. We hypothesized dexmedetomidine added to intravenous patient-controlled analgesia (PCA) could enhance the quality of recovery (QoR) in patients undergoing laparotomy surgery. ABSTRACT.METHODS: In this randomized, double-blind, controlled study, 100 patients undergoing laparotomy surgery were randomly allocated into two groups: Dexmedetomidine (group D) and control (group S). Patients in the group D (n = 46) received dexmedetomidine 0.04 ug/(kg·h) plus sufentanil 0.02 ug/(kg·h) for 48 h after laparotomy surgery, and in the group S (n = 47) received sufentanil 0.04 ug/(kg·h) only. The QoR-15 scores, postoperative pain, rescue analgesia, recovery of gastrointestinal function, patient satisfaction and adverse effects were recorded. ABSTRACT.RESULTS: The QoR-15 scores were significantly higher in the group D than in the group S on postoperative day (POD) 1, 2, 3 and 5 (P < 0.05). The visual analog scale (VAS) scores were significantly lower in the group D compared with group S within 48 h after surgery (P < 0.05). The pressing times of analgesic pump and rescue tramadol were not significantly different between the two groups (P > 0.05). The incidence of nausea was significantly lower in the Group D. No hypotension, bradycardia, or respiratory depression was observed. ABSTRACT.CONCLUSIONS: The addition of dexmedetomidine to PCA enhanced patient-centered recovery, reduced pain and adverse effect, and improved patient satisfaction after laparotomy surgery. BODY.INTRODUCTION: Laparotomy, one of the most common surgical procedures, is widely used in clinical practice, which is the preferred choice for abdominal sepsis and abdominal compartment syndrome [1, 2]. Laparotomy would cause great damage, associated with a high incidence of postoperative pain, increase the incidence of complications and thus delay the process of postoperative recovery [3, 4]. Promoting early recovery has important clinical significance, which is one of the most important medical tasks. Dexmedetomidine, a highly selective α2-adrenoceptor activation, is a sedative, analgesic, pathologic anxiety relieving, and anti-inflammatory drug, without respiratory depression and opioid-sparing effect [5-7]. Dexmedetomidine is effective, alone or in combination with other analgesics, in reducing postoperative pain [8-10]. Furthermore, dexmedetomidine has been shown to decrease nausea and vomiting, improve mood and speed up patient recovery in a variety of medical and surgical patients [8, 9]. It is becoming increasingly important to measure the quality of recovery (QoR) from the perspective of the patient. Most recent studies focused on recovery time, pain, or other adverse reactions, however, these are not enough to reflect the recovery of the patient from anesthesia and surgery. The QoR-15, which is a patient-centered QoR measure, can effectively evaluate the quality of postoperative rehabilitation [11, 12]. We hypothesized that intravenous patient-controlled analgesia (PCA) with dexmedetomidine would beneficially affect patient-centered QoR and that several early clinical recovery variables during hospitalization, such as pain, nausea, and patient satisfaction. BODY.RESULTS: BODY.DEMOGRAPHIC DATA AND SURGERY/ANESTHESIA-RELATED INFORMATION: Between December 2016 and May 2017, of 427 patients screened, a total of 100 patients were enrolled. With 7 patients excluded, 93 (93.0%) patients were included in the statistical analysis: 46 patients in group D and 47 patients in group S (Figure 1). There were no significant difference between the two groups in patient characteristics and intraoperative variables were similar (P > 0.05; Table 1). Figure 1BODY.FLOW DIAGRAM OF THE STUDY: Table 1BODY.PATIENT CHARACTERISTICS AND INTRAOPERATIVE DATA: Group S(n=47)Group D(n=46)Difference (95% CI)P ValueSex, male:female 27 (57.4%):20 (42.6%) 24 (52.2%):22 (47.8%) 0.609 Age, yr 54.6 ± 14.6 51.2 ± 15.1 3.3 (-2.8 to 9.4) 0.290 Height, cm 167.4 ± 6.2 166.4 ± 6.9 1.1 (-1.7 to 3.8) 0.442 Weight, kg 63.4 ± 8.8 62.8 ± 9.2 0.6 (-3.1 to 4.3) 0.757 ASA physical status 2 (1-3) 2 (1-3) 0 (0 to 0) 0.866 Surgical site 0.650  Gastrointestinal disease 23 (48.9%) 19 (41.3%) 0.460  Hepatobilitary diseases 18 (38.3%) 22 (47.8%) 0.406  Pancreatic diseases 6 (12.8%) 5 (10.9%) 0.777 Intraoperative data  Anesthesia time, h 4.3 ± 1.0 4.2 ± 1.0 0.1 (-0.3 to 0.5) 0.773  Sufentanil usage, ug 35.7 ± 6.7 34.6 ± 6.2 1.1 (-1.5 to 3.8) 0.408  Remifentanil usage, ug 1357.4 ± 519.3 1338.2 ± 503.4 19.2 (-191.5 to 229.9) 0.857  Time to extubation, min 10.8 ± 4.4 10.9 ± 4.3 -0.2 (-2.0 to 1.6) 0.852 Notes: Data are number of patients (%), median (range) or median ± standard deviation. Abbreviation: CI, confidence interval. BODY.QUALITY OF RECOVERY: Baseline QoR-15 scores measured preoperatively did not differ between the two groups (Table 2, Figure 2). The QoR-15 scores were lowest on POD 1 in both groups. The QoR-15 scores were significantly higher in the group D than in the group S on POD 1, 2, 3 and 5 (99.7 ± 6.9 vs 92.5 ± 6.4, 112.3 ± 6.9 vs 106.8 ± 8.5, 116.0 ± 7.8 vs 111.1 ± 8.0, 121.9 ± 5.2 vs 116.7 ± 7.7, respectively. Figure 2), but still lower than their baseline. The dimensions of emotional state, physical comfort and pain were significantly improved in the group D (P < 0.05; Table 2). There is no significant difference between the two groups of psychological support and physical independence. Table 2BODY.QUALITY OF RECOVERY (QOR-15) DIMENSIONS AND SCORES: Group S(n=47)Group D(n=46)Difference (95% CI)P ValueQoR-15 dimensions  Emotional state   Preoperative 37.4 ± 1.5 37.2 ± 1.9 0.2 (-0.5 to 0.9) 0.596   POD 1 25.8 ± 3.4 28.8 ± 5.3 -3.0 (-4.9 to -1.2) 0.001**   POD 2 34.1 ± 3.0 35.3 ± 1.6 -1.2 (-2.2 to -0.2) 0.018*   POD 3 34.4 ± 4.1 36.0 ± 2.5 -1.6 (-3.0 to -0.2) 0.027*   POD 5 35.3 ± 2.1 36.7 ± 1.5 -1.4 (-2.2 to -0.6) 0.000**   POD 7 36.9 ± 1.0 36.5 ± 1.4 0.4 (-0.1 to 0.9) 0.083  Physical comfort   Preoperative 41.5 ± 2.2 40.7 ± 2.4 0.8 (-0.1 to 1.8) 0.088   POD 1 35.7 ± 5.6 38.0 ± 4.5 -2.3 (-4.3 to -0.2) 0.034*   POD 2 35.8 ± 5.4 38.5 ±5.4 -2.7 (-4.9 to -0.4) 0.019*   POD 3 37.2 ± 4.9 39.5 ± 5.0 -2.3 (-4.3 to -0.2) 0.030*   POD 5 37.7 ± 6.0 40.3 ± 4.1 -2.6 (-4.8 to -0.5) 0.016*   POD 7 41.2 ± 2.4 40.3 ± 2.5 0.9 (-0.1 to 1.9) 0.084  Psychological support   Preoperative 19.2 ± 0.4 19.1 ± 0.3 0.04 (-0.11 to 0.19) 0.596   POD 1 18.5 ± 0.7 18.6 ± 0.7 -0.1 (-0.4 to 0.2) 0.514   POD 2 18.6 ± 1.9 18.6 ± 2.8 0.1 (-0.9 to 1.1) 0.883   POD 3 18.8 ± 1.4 18.9 ± 0.3 -0.1 (-0.5 to 0.3) 0.692   POD 5 19.2 ± 0.4 19.1 ± 0.4 0.1 (-0.1 to 0.2) 0.468   POD 7 19.1 ± 0.3 19.0 ± 0.1 0.08 (-0.02 to 0.19) 0.098  Physical independence   preoperative 16.1 ± 0.4 16.3 ± 0.5 -0.18 (-0.36 to 0.01) 0.064   POD 1 1.1 ± 0.2 1.1 ± 0.3 -0.07 (-0.19 to 0.06) 0.284   POD 2 1.4 ± 1.2 1.5 ± 1.5 -0.1 (-0.7 to 0.4) 0.673   POD 3 2.2 ± 2.1 2.5 ± 2.1 -0.2 (-1.1 to 0.6) 0.610   POD 5 5.9 ± 2.3 6.3 ± 1.8 -0.4 (-1.3 to 0.5) 0.345   POD 7 8.0 ± 4.2 8.8 ± 1.6 -0.8 (-2.1 to 0.5) 0.228  Pain   Preoperative 19.8 ± 0.5 19.6 ± 0.8 0.2 (-0.1 to 0.5) 0.166   POD 1 11.5 ± 2.3 13.2 ± 3.2 -1.8 (-2.9 to -0.6) 0.003**   POD 2 16.9 ± 3.1 18.5 ± 2.3 -1.5 (-2.7 to -0.4) 0.007**   POD 3 18.5 ± 1.7 19.2 ±1.5 -0.69 (-1.34 to -0.03) 0.041*   POD 5 18.6 ± 2.2 19.5 ± 1.1 -0.8 (-1.6 to -0.1) 0.020*   POD 7 19.7 ± 0.9 19.6 ± 1.0 0.1 (-0.3 to 0.5) 0.562 Notes: Data are median ± standard deviation. Abbreviation: CI, confidence interval. * P < 0.05, group S vs. group D, ** P < 0.01, group S vs. group D. Figure 2BODY.THE QOR-15 SCORES ON PREOPERATION, POD1, POD2, POD3, POD5 AND POD7: Abbreviation: POD, postoperative day. ** P < 0.01, group S vs. group D. BODY.POSTANESTHESIA CARE UNIT INFORMATION: The incidence of nausea in PACU was significantly lower in the group D than in the group S (15.2% vs 36.2%; Table 3). While, the incidence of vomiting and antiemetic drug administered had no difference between the two groups. The VASR was significantly lower in the group D than in the group S (1.4 ± 2.7 vs 1.5 ± 2.0; Figure 3). While, the rescue sufentanil had no significant difference between the two groups (Table 3 Table 3BODY.POSTANESTHESIA CARE UNIT PARAMETERS: Group S(n=47)Group D(n=46)Difference (95% CI)P ValueNausea 17 (36.2%) 7 (15.2%) 0.021* Vomiting 3 (6.4%) 1 (2.2%) 0.617 Administered antiemetic drug 5 (10.6%) 2 (4.3%) 0.435 Administered rescuesufentanil 28 (59.6%) 20 (43.5%) 0.120 Duration in PACU, min 60.6 ± 22.4 67.9 ± 31.6 -7.3 (-18.6 to 4.0) 0.203 Patient satisfaction 2.8 ± 0.8 3.2 ± 0.8 -0.4 (-0.8 to -0.1) 0.012* Notes: Data are number of patients (%) or median ± standard deviation. Abbreviation: CI, confidence interval. * P < 0.05, group S vs. group D. Figure 3Postoperative pain at rest (A), and at movement (B). Abbreviation: VAS, visual analogue scale (VAS; with 0, no pain, to 10, the worst imaginable pain). * P<0.05, group S vs. group D, ** P < 0.01, group S vs. group D. BODY.ANALGESIC EFFECT EVALUATION: Postoperative pain was assessed with the visual analogue scale (VAS; with 0, no pain, to 10, the worst imaginable pain). The VAS scores were lower in the group D compared with group S within 48 h after surgery (P < 0.05; Figure 3). While, pressing times of analgesic pump and rescue tramadol used had no significant difference between the two groups (P < 0.05; Table 4). Table 4BODY.THE PCA BUTTON PUSHED AND RESCUE TRAMADOL REQUIRED: Group S(n=47)Group D(n=46)P ValueButton pushed on 2h, n (%)  0/≥1/≥3 25(53.2%)/21(44.7%)/1(2.1%) 26(56.5%)/19(41.3%)/1(2.2%) 0.916 Button pushed on 8h, n (%)  0/≥1/≥3 24(51.1%)/19(40.4%)/4(8.5%) 24(52.2%)/17(37.0%)/5(10.9%) 0.911 Button pushed on 12h, n (%)  0/≥1/≥3 30(63.8%)/14(29.8%)/3(6.4%) 33(71.7%)/12(26.1%)/1(2.2%) 0.533 Button pushed on POD 1, n (%)  0/≥1/≥3 30(63.8%)/13(27.7%)/4(8.5%) 31(67.4%)/10(21.7%)/5(10.9%) 0.808 Button pushed on POD 2, n (%)  0/≥1/≥3 33(70.2%)/10(21.3%)/4(8.5%) 34(73.9%)/10(21.7%)/2(4.3%) 0.821 Rescue tramadol on 2h, n (%)  0/≥1/≥2 32(68.1%)/12(25.5%)/3(6.4%) 33(71.7%)/12(26.1%)/1(2.2%) 0.750 Rescue tramadol on 8h, n (%)  0/≥1/≥2 32(68.1%)/13(27.7%)/2(4.3%) 30(65.2%)/14(30.4%)/2(4.3%) 0.931 Rescue tramadol on 12h, n (%)  0/≥1/≥2 37(78.7%)/9(19.1%)/1(2.1%) 37(80.4%)/7(15.2%)/2(4.3%) 0.836 Rescue tramadol on POD 1, n (%)  0/≥1/≥2 38(80.9%)/7(14.9%)/2(4.3%) 33(71.7%)/10(21.7%)/3(6.5%) 0.586 Rescue tramadol on POD 2, n (%)  0/≥1/≥2 44(93.6%)/3(6.4%)/0(0.0%) 42(91.3%)/3(6.5%)/1(2.2%) 0.837 Rescue tramadol on POD 3, n (%)  0/≥1 44(93.6%)/3(6.4%) 43(93.5%)/3(6.5%) 1.000 Rescue tramadol on POD 5, n (%)  0/≥1 44(93.6%)/3(6.4%) 44(95.7%)/2(4.3%) 1.000 Rescue tramadol on POD 7, n (%)  0/≥1 46(97.9%)/1(2.1%) 44(95.7%)/2(4.3%) 0.617 Notes: Data are number of patients (%). BODY.FLATUS AND SATISFACTION: The time to first flatus after surgery was shorter in the group D than in the group S (p < 0.05; Figure 4). The scores of satisfaction of group D were higher than that of group S (p < 0.05). Figure 4Results of time to first flatus after operation (A), and patient satisfaction (B). Abbreviation: PACU, postanesthesia care unit; POD, postoperative day. * P<0.05, group S vs. group D, ** P < 0.01, group S vs. group D. BODY.POSTOPERATIVE ADVERSE EFFECTS: There were no differences between the two groups in the incidence of postoperative adverse effects with the exception of reduced incidence of nausea within 48 h after surgery in the group D (P < 0.05; Table 5). Table 5BODY.ADVERSE EFFECTS: Group S(n=47)Group D(n=46)P ValueNausea, n (%)  0-8H 21 (44.7%) 9 (19.6%) 0.010*  0-24H 25 (53.2%) 12 (26.1%) 0.008**  0-48H 26 (55.3%) 14 (30.4%) 0.015* Vomiting, n (%)  0-8H 3 (6.4%) 1 (2.2%) 0.617  0-24H 4 (8.5%) 2 (4.3%) 0.677  0-48H 4 (8.5%) 2 (4.3%) 0.677 Notes: Data are number of patients (%). * P < 0.05, group S vs. group D, ** P < 0.01, group S vs. group D. BODY.DISCUSSION: Kehlet is the first one to put forward the concept of Enhanced Recovery After Surgery (ERAS) in 2001 to emphasize earlier recovery after operations [14]. ERAS programs can reduce the rate of surgical complications, reduce hospital costs and increase patient satisfaction [15, 16]. In our study, patient-centered recovery was enhanced significantly with the addition of dexmedetomidine to PCA. Furthermore, dexmedetomidine reduced the incidence of nausea, pain, adverse effect, and improved patient satisfaction after laparotomy surgery. There is a variety of assessments of QoR in clinical practice, which has become an important outcome of research [17-19]. Patient-centered QoR is superior to other assessments in postoperative period, which can be more intuitively and accurately reflect the patient’s recovery [20]. The QoR-15, patient-centered, evolved from QoR-40, is a 15-item scoring system, an 11-point numerical rating scale (for negative items, 0 = “all of the time” to 10 = “none of the time”; for positive items, the scoring was reversed; score range from 0 to 150), including 5 demensions: physical comfort, physical independence, psychological support, emotional state and pain [12]. QoR-15 can effectively and extensively evaluate postoperative QoR, which can be completed within 3 min and is comparable to the more detailed scale QoR-40 [12, 20]. Dexmedetomidine, as a selective α2-adrenoreceptor agonist, has the characteristics of sedation, anxiolysis, analgesia, and sympatholysis via receptors in locus ceruleus and spinal cord without significant respiratory depression [21, 22]. Studies have reported that dexmedetomidine contributes to early postoperative recovery in the various kinds of surgery including bariatric surgery, thoracic surgery, gynecological laparoscopic surgery, abdominal hysterectomy surgery, abdominal colectomy, thyroidectomy surgery, vertebralsurgery, nasal surgery mastectomy surgery and so on [23-28]. A single-item satisfaction assessment, however, is poorly reliable and is not sufficient to assess postoperative recovery [29, 30]. The QoR-15 scoring system was applied in our study to evaluate the QoR after operation. The scores of QoR-15 were higher in the group D than in the group S. The dimensions of pain, emotional state, and physical comfort were significantly improved in the group D. Furthermore, the scores of patient satisfaction to the early recovery process were significantly higher when dexmedetomidine was used. The effect of dexmedetomidine on postoperative pain remains controversial. A prospective randomized controlled trial by Cheng et al, evaluated 59 patients who received dexmedetomidine, and reported a significant reduction in pain scores after abdominal operations compared with control group. Other randomized investigations, however, reported no significant difference in postoperative pain [31, 32]. In some clinical studies, intravenous administration of dexmedetomidine presents a postoperative opioid-sparing effect with no reduction in postoperative pain [32]. In the present study, the administration of dexmedetomidine plus sufentanil PCA significantly improved the dimension of pain scores of QoR-15. Furthermore, postoperative VAS pain scores were lower as well. For pressing times of analgesic pump and supplemental requiremanent for tramadol, there were no significant difference between the two groups. However, in the group D, the PCA concentration of sufentanil were half that of the group S. Dexmedetomidine has an anti-nociception effect on skin and visceral pain, which can be reversed by naloxone pretreatment, indicating a possible interaction through opioid systems [33, 34]. A reduction in postoperative PCA opioid requirements may be attributed to the enhanced effect of dexmedetomidine on opioid analgesia [35, 36]. Our results confirmed opioid-sparing effect of dexmedetomidine. Anxiety is one of the main causes influencing postoperative recovery [37]. In order to enhance recovery and discharge, postoperative physical and psychological stress therapy should be given attention [38]. Studies have indicated that dexmedetomidine provides excellent effect of sedation without respiratory depression [5, 39-41]. In our study, dexmedetomidine beneficially affected the postoperative emotional state (assessed on the QoR-15 dimension of emotion), which was consistent with previously published studies. The improved emotional state in the group D may be induced by the effect of dexmedetomidine on the central nervous system [40]. Dexmedetomidine has the property of anti-inflammatory effect as well, which may contribute to improving emotional state [42, 43]. Furthermore, the analgesic effect of dexmedetomidine can help relieve anxiety as well [5, 44]. The dimension of physical comfort of QoR-15 primarily including nausea and vomiting, sleeping and appetite. In our study, as expected, we observed that the scores in the part of physical comfort of QoR-15 significantly improved in the patients who were administered dexmedetomidine. Previous studies have showed a decrease in the incidence of postoperative nausea and vomiting [45, 46]. In the present study, with consistent to previous clinical trials, the incidence of nausea after operation were reduced in the group D, which may contribute to improve physical comfort of patients. It has been reported that dexmedetomidine has a positive effect on the quality of postoperative sleep without respiratory depression [40, 47]. In our study, the sleep quality was improved in the group D, which could improve the comfortable degree of patients. Surgery has an adverse effect on the movement of the gastrointestinal tract, leading to decreased appetite [48]. In our study, patients administered dexmedetomidine presented a better appetite. Furthermore, the time to first flatus after operation is shorter in the group D, which could promote early recovery of patients. There are some limitations in our study. First, there is no consensus on the optimal dose of dexmedetomidine contributed to postoperative recovery. The speed of PCA dexmedetomidine at 0.4 ug/(kg·h) is derived from a previous study about abdominal total hysterectomy [46]. Future dose-related studies are needed to establish an optimal dose of dexmedetomidine for early postoperative rehabilitation. Second, all patients in this study used antiemetic drug conventionally, which may affect the antiemetic effect of dexmedetomidine. However, we still observed that the incidence of nausea in the group D was lower than the group S. Third, we did not record the cumulative amount of PCA sufentanil and the rescue tramadol. However, we recorded the frequency of PCA bottom pushed and rescue tramadol used. Finally, studies have demonstrated that dexmedetomidine has a few adverse reactions [31, 49]. In present study, we did not detect a difference in dexmedetomidine-related adverse effects, which is probably related to the low dose of dexmedetomidine. Many clinical researches have showed that small-dose dexmedetomidine infusion resulted in reversible sedation, mild analgesia, reducing the incidence of nausea and vomiting, without inducing adverse effect [50, 51]. In summary, the administration of dexmedetomidine significantly enhanced patient-centered postoperative QoR. The incidence of nausea after operation was reduced, the quality of sleep was improved and that a faster recovery of gastrointestinal function accompanied by a better appetite when dexmedetomidine was administered. Furthermore, the scores of patient satisfaction to the early recovery process were higher with a better control of pain. We recommend the use of dexmedetomidine as an important adjunct to postoperative PCA to improve patient-centered QoR after laparotomy surgery. BODY.MATERIALS AND METHODS: BODY.PATIENTS AND STUDY DESIGN: This randomized, double-blind, controlled study was approved by the Ethical Committee of West China Hospital and was registered in the Chinese Clinical Trial Registry (ChiCTR-IPR-16010184). This trial was performed following the Declaration of Helsinki and obtained written informed consent from all subjects before participating in the study. Patients aged 18-80 years, ASA I to III, scheduled to laparotomy surgery in West China Hospital of Sichuan University, China, between December 2016 and May 2017 were included. Exclusion criteria: (1) patients with atrioventricular block, sinus bradycardia or other serious heart disease; (2) patients with body mass index >30 kg/m2; (3) patients with allergic to the medications used; (4) patients with long history of taking analgesics or antidepressants; (5) patients who had taken other test drugs within three months prior to the study or were involved in other clinical trials; (6) patients who were pregnant or breastfeeding; (7) patients who could not cooperate or refused; (8) patients who were admitted to Intensive Care Unit (ICU) after surgery. Finally, 100 patients were enrolled. Patients were randomly assigned to the group D (n=50) or group S (n=50) by a computer-generated randomization table. Before the experiment, a total of 100 random numbers were generated according to the 1: 1 ratio of the two groups The grouping was sealed into the sealed envelopes, kept by the operating room pharmacy, which was responsible for the preparation of the study medication. The storage bag of the PCA contained 100 ml solution with the rate of 2 ml/h background infusion, a bolus dose of 0.5 ml and a lock time of 15 min for 48 h after surgery. In the group D, the PCA contained dexmedetomidine and sufentanil, with the infusion rate of 0.04 ug/(kg·h) and 0.02 ug/(kg·h). In the group S, the PCA contained sufentanil only, with the infusion rate of 0.04 ug/(kg·h). The PCA was used to achieve the pain score at rest < 4. All the surgeons, anesthesiologists, nurses, patients and researchers were blinded to the group assignment. BODY.ANESTHETIC AND SURGICAL MANAGEMENT: Before the surgery, patients were informed about the use of the PCA system. Once entering in the operating room, patients were routinely monitored of five-lead electrocardiogram (ECG), pulse oxygen saturation (SPO2), noninvasive blood pressure (BP) and established venous access. Followed by intravenous injection of propofol 2 mg/kg, sufentanil 0.3 ug/kg, and cisatracurium 0.2 mg/kg, endotracheal intubation was performed. Ohmenda-Datex Model 7100 Anesthesia Machine was used for mechanical ventilation (airway peak pressure not more than 40 cmH2O, the oxygen saturation maintained ≥95%, the end-tidal carbon dioxide partial pressure (ETCO2) maintained between 35-45 mmHg). Anesthesia was maintained by inhalation of 1-3% sevoflurane, infusion of remifentanil (0.1-0.2) ug/(kg·min), intermittent administration of cisatracurium and sufentanil to maintain bispectral index (BIS) between 40 and 60. According to the amount of surgical bleeding and blood pressure to adjust the infusion rate and the use of vasoactive drugs, according to the current blood transfusion guidelines to determine the input of blood products. Once started closing the abdominal cavity, PCA system was started and cisatracurium and sufentanil were discontinued. At the same time, ondansctron o.1 mg/kg and tramadol 1.5 mg/kg were administered to prevent postoperative pain, nausea and vomiting. The administration of sevoflurane and remifentanil were discontinued 5 min before the ending of the operation. When the operation was completed, oxygen flow was increased up to 6 L/min in order to quickly wash out sevoflurane. When the patient’s spontaneous breathing tidal volume reached 3 ml/kg, neostigmine 0.04 mg/kg and atropine 0.02 mg/kg were administrated to reverse neuromuscular relaxation. Patients were extubated after recovery from anesthesia, and then transferred to the postanesthesia care unit (PACU). Once entering the PACU, patients were evaluated the intensity of pain using visual analog scale (VAS), the incidence of bradycardia, shiver, nausea and vomiting every 5 minutes. If the VAS at rest (VASR) ≥ 4, the PCA button was pressed. If the pain still could not be relieved or the VASR ≥ 7, the rescue analgesia of sufentanil 0.1 ug/kg was administrated every 5min until VASR was less than 4. Patients were transferred to ward when Modified Aldrete score ≥ 9. BODY.DATA COLLECTION: Our primary outcome was the score of QoR-15 on postoperative day (POD) 3. The QoR-15 questionnaire was conducted on preoperative, POD 1, 2, 3, 5 and 7. The intensity of pain, the rescue analgesics, the incidence of adverse events including bradycardia (HR < 50 beats/min), hypotension (MBP was reduced 20% from the baseline), shiver, nausea and vomiting were evaluated at 2, 8 and 12 h after operation and POD 1, 2, 3, 5 and 7. If the VASR ≥ 4, during 48 h after surgery, the PCA button was pressed. If the pain still could not be relieved, tramadol 100 mg was used intravenous every 30min until VASR was less than 4. If serious adverse events occurred, immediately stop using PCA, and appropriately treated adverse reactions. The time of discharging from PACU, time to first flatus after surgery and the score of patient satisfaction evaluated by a 5-point scale (1, very dissatisfied; 2, not satisfied; 3, neither dissatisfied nor satisfied; 4, satisfied; 5 very satisfied) were also recorded. BODY.STATISTICAL ANALYSIS: The sample size of this study was based on the score of QoR-15 in the 72h after surgery. The score of QoR-15 in the 72h after surgery was 122 (SD 24), which was based on a previous study [13

TITLE: Total Laparoscopic Restorative Proctocolectomy: Are There Advantages Compared with the Open and Hand-Assisted Approaches? ABSTRACT.PURPOSE: A randomized, controlled trial comparing hand-assisted laparoscopic restorative proctocolectomy with open surgery did not show an advantage for the laparoscopic approach. The trial was criticized because hand-assisted laparoscopic restorative proctocolectomy was not considered a true laparoscopic proctocolectomy. The objective of the present study was to assess whether total laparoscopic restorative proctocolectomy has advantages over hand-assisted laparoscopic restorative proctocolectomy with respect to early recovery. ABSTRACT.METHODS: Thirty-five patients underwent total laparoscopic restorative proctocolectomy and were compared to 60 patients from a previously conducted randomized, controlled trial comparing hand-assisted laparoscopic restorative proctocolectomy and open restorative proctocolectomy. End points included operating time, conversion rate, reoperation rate, hospital stay, morbidity, quality of life, and costs. The Medical Outcomes Study Short Form 36 and the Gastrointestinal Quality of Life Index were used to evaluate general and bowel-related quality of life. ABSTRACT.RESULTS: Groups were comparable for patient characteristics, such as sex, body mass index, preoperative disease duration, and age. There were neither conversions nor intraoperative complications. Median operating time was longer in the total laparoscopic compared with the hand-assisted laparoscopic group (298 vs. 214 minutes; P < 0.001). Morbidity and reoperation rates in the total laparoscopic, hand-assisted laparoscopic, and open groups were comparable (29 vs. 20 vs. 23 percent and 17 vs.10 vs. 13 percent, respectively). Median hospital-stay was 9 days in the total laparoscopic group compared with 10 days in the hand-assisted laparoscopic group and 11 days in the open group (P = not significant). There were no differences in quality of life and total costs. ABSTRACT.CONCLUSIONS: There were no significant short-term benefits for total laparoscopic compared with hand-assisted laparoscopic restorative proctocolectomy with respect to early morbidity, operating time, quality of life, costs, and hospital stay. Restorative proctocolectomy is considered the operation of choice for patients with ulcerative colitis (UC) and familial polyposis coli (FAP). Several studies have demonstrated laparoscopic approaches for restorative proctocolectomy to be feasible and safe. However, most of these studies concluded that recovery after laparoscopic restorative proctocolectomy (LRP) was similar or only marginally improved compared with conventional open restorative proctocolectomy (ORP).1–3 The only randomized trial comparing LRP with ORP, applying a hand-assisted laparoscopic technique (HAL-RP), indicated that there were no benefits with respect to early recovery and morbidity.4 Critics argued that the hand-assisted laparoscopic technique could not be considered a true laparoscopic procedure. It was suggested that the lack of difference in outcome between HAL-RP and ORP could be explained by the fact that the HAL-RP is a hybrid laparoscopic procedure consisting of a hand-assisted colectomy followed by an open restorative proctectomy via the Pfannenstiel incision used for the insertion of the handport. The open proctectomy during HAL-RP requires the use of a ring-retractor, which causes considerable strain on the abdominal wound. The question emerged whether a pure laparoscopic restorative proctocolectomy would show the expected benefits. Therefore, the authors decided to adapt a total laparoscopic approach by combining a total laparoscopic colectomy and laparoscopic restorative proctectomy (TLRP) on a cohort of patients and compare outcomes with those after HAL-RP and ORP. TLRP is believed to be less invasive than the hand-assisted laparoscopic approach because of lesser manipulation of the bowel and abdominal wound. Although theoretic advantages of both procedures are strongly debated in the literature, the advantages of either procedure cannot be sustained by good quality studies comparing both approaches directly. The objective of this study was to determine whether a total laparoscopic approach for restorative proctocolectomy has short-term advantages compared with a hand-assisted laparoscopic approach with respect to morbidity, early recovery, cost, and quality of life. Because no significant differences between HAL-RP and ORP were found in the previously conducted RCT, the results after TLRP will be compared with those after ORP as well. BODY.PATIENTS AND METHODS: In the period April 2004 to March 2006, patients eligible for restorative proctocolectomy were operated by a total laparoscopic approach and prospectively evaluated. Short-term outcomes were compared with those of a patient population of a previously conducted, randomized, controlled two-center trial. In this trial, conducted in the period January 2000 to August 2003, patients were allocated to HAL-RP or ORP. Short-term results (perioperative data and quality of life (QOL) until 3 months after operation) of this randomized trial have been published previously.5 The surgical technique of TLRP consisted of a medial to lateral total laparoscopic proctocolectomy using a 6-trocar technique. After complete laparoscopic dissection of the rectum down to the pelvic floor, the midrectum is transected laparoscopically by using a linear endostapler with knife (Endopath®, TSB45, Ethicon Endo-Surgery, Amersfoort, The Netherlands). The colon and half of the rectum can now be extracted through a 6-cm Pfannenstiel incision. With the colon out of the way, the rectum stump can be retracted with a Satinsky clamp and cross stapled 1-cm to 2-cm proximal to the dentate line by using an open TL 30 stapler (Proximate® TL30, Ethicon Endo-Surgery) to ensure a transverse low rectal cross-stapling. The terminal ileum was exteriorized and the pouch was created by using a 100-mm linear cutter (Proximate TLC10®, Ethicon Endo-Surgery) and the anvil of the circular stapler (Proximate CDH29®, Ethicon Endo-Surgery) was inserted in the base of the pouch. After re-establishing the pneumoperitoneum, the ileoanal anastomosis was created laparoscopically. The surgical technique of HAL-RP has been described previously.6 In summary, an 8-cm Pfannenstiel incision was made at the start of the operation. After mobilizing the sigmoid through this incision, the handport was placed in the Pfannenstiel incision. Three additional trocars were inserted. Under manual guidance, mobilization of the large bowel was performed. Proctectomy was performed openly via the Pfannenstiel incision. A J-pouch was constructed for both the total laparoscopic, hand-assisted laparoscopic, and open approaches. A defunctioning ileostomy in all groups was only given in selected cases: in case of active inflammatory disease defined as bloody stool and high-dosage corticosteroids (>10 mg/day), in case of difficult dissection or technical difficulties during construction of the anastomosis, in case of bleeding, or in case of an incomplete donut. Postoperative care of the TLRP group was identical to the patient groups who underwent HAL-RP and ORP. All patients who did not have a defunctioning ileostomy received a pouch catheter, which was never removed until the sixth day after surgery. Oral intake was advanced to liquids and nutritional supplements as fast as possible after surgery. Only after removal of the pouch catheter, patients were allowed to have solids because of fear of solid stool blocking the pouch catheter. In patients who had a defunctioning ileostomy, oral intake was advanced as fast as possible to a normal diet. Discharge criteria in the three groups were equal and consisted of 1) adequate pain control with oral drugs, 2) absence of nausea, 3) passage of first flatus and/or stool, as well as an acceptable stool frequency (<10/day), 4) ability to tolerate solid food, 5) mobilization and self support as preoperative, and 6) acceptance of discharge by the patient. Primary end points were operating time, conversion rate, early morbidity (within 30 days after surgery), morphine requirement, and costs. Daily (postoperative Day (POD) 1, POD2, and POD3) and total morphine requirement was assessed in all patients who received patient-controlled analgesia (PCA). The allocation to PCA or epidural analgesia was at the discretion of the attending anesthesiologist and patient’s preference. The calculation of total costs was based on the amounts for costs for material used during the procedure, costs for use of an operating room with personnel, costs for relaparotomies or relaparoscopies, and costs for admission days. Preoperative costs were not taken into account. Secondary end points were time to resumption of full liquid (>1,000 ml) and normal (solid) diet, and QOL measured by the Medical Outcomes Study Short Form 36 (SF-36) and the Gastrointestinal Quality of Life Index (GIQLI) preoperatively, and at one, two, and four weeks, and three months after the operation.7,8 BODY.STATISTICS: All data are presented as median and range, unless otherwise specified. The nonparametric Kruskal-Wallis test was used to compare discrete and continuous variables between the three groups. If P < 0.05, a post-hoc analysis using the Mann-Whitney U test was performed to compare discrete and continuous data between two groups. The chi-squared test, or Fisher’s exact test when appropriate were used to compare categorical or dichotomous variables between groups. For comparison of results of different time points of QOL, a repeated measures multivariate ANOVA procedure was used. BODY.RESULTS: In the study period, 35 of 37 patients with UC or FAP eligible for surgery had a one-stage TLRP. Patient characteristics of the three groups are shown at Table 1. There were no significant differences between the groups, although patients from the HAL-RP group tended to be younger than patients from both other groups and the proportion of patients with FAP in the TLRP group tended to be smaller than in the HAL-RP and ORP group. As a consequence, the number of patients receiving immunosuppressive drugs in the TLRP was higher compared with both other groups. Table 1Characteristics of the 95 patients before one-stage restorative proctocolectomy  TLRP (n = 35)HAL-RP (n = 30)ORP (n = 30)P value*P value†P value‡Male:female ratio 11:24 9:21 15:15 0.194 — — Age (yr) 36 (15–54) 29 (16–57) 35 (16–57) 0.045 0.076 0.43 UC:FAP ratio 30:5 20:10 20:10 0.13 — — BMI (kg/m2) 24.3 (16.6–39.5) 22.6 (18.1–34.7) 23.3 (17.2–34.2) 0.692 — — Duration of disease (yr) 7 (0.1–30) 6 (0.5–37) 4 (0.1–15) 0.345 — — TLRP = total laparosopic restorative proctocolectomy; HAL-RP = hand-assisted laparoscopic restorative proctocolectomy; ORP = open restorative proctocolectomy; M = male; F = female; UC = ulcerative colitis; FAP = familial polyposis coli; BMI = body mass index. Data are numbers of patients or medians with ranges in parentheses unless otherwise indicated. *TLRP vs. HAL-RP vs. ORP (Kruskal-Wallis for continuous variables, chi-squared test, or Fisher’s exact test (n < 5) for categorical variables). †TLRP vs. HAL-RP (Mann-Whitney U test for continuous variables, chi-squared test, or Fisher’s exact test (n < 5) for categorical variables). ‡TLRP vs. ORP (Mann-Whitney U test for continuous variables, chi-squared test, or Fisher’s exact test (n < 5) for categorical variables). There were no intraoperative complications in either group and there were no conversions in the laparoscopic groups (Table 2). Operating time was significantly longer in the TLRP compared with the HAL-RP and ORP groups. Table 2Results of peri-operative outcome parameters after TLRP vs. HAL-RP and ORP  TLRP (n = 35)HAL-RP (n = 30)ORP (n = 30)P value§P value*P value**Operating time‡ (min) 298 (235–375) 214 (149–400) 133 (97–260) 0.000 0.000 0.000 Primary protecting loop ileostomy 12 8 7 0.601 — — Hospital stay‡ 9 (5–39) 10 (5–31) 11 (6–28) 0.139 — — Minor complications*** 3 1 3 0.698 — —  Wound infection 1 1 1  Urinary tract infection 2 0 1  Pneumonia 0 0 1 Major complications*** §§ 7 5 4 0.745 — —  Persistent ileus/inability to defecate 1 (1) 3 (1) 2 (2)  Anastomotic leakage 2 (2) 2 (2) 1 (1)  Intra-abdominal abscess 1 (1) 0 0  SB herniation through omentum 1 (1) 0 0  Stoma necrosis 1 (1) 0 0  Pulmonary embolism 1 0 0  Corpus alienum 0 0 1 (1) Total number of patients with re-operation 6 3 4 0.705 — — All patients with diversion (including those after re-operation) 16 11 11 0.686 — — ‡ Median with range § P TLRP vs HAL-RP vs ORP (Kruskall Wallis for continues variables, Chi square test or Fisher’s exact test (n < 5) for categorical variables) * P TLRP vs HAL-RP (Mann Whitney U test for continues variables, Chi square test or Fisher’s exact test (n < 5) for categorical variables) **P TLRP vs ORP (Mann Whitney U test for continues variables, Chi square test or Fisher´s exact test (n<5) for categorical variables) *** Within 30 days after operation SB: small bowel §§ numbers between parantheses are the numbers of patients requiring a re-operation for specific complication TLRP: Total laparosopic restorative protocolectomy HAL-RP: Hand-assisted laparoscopic restorative proctocolectomy ORP: Open restorative proctocolectomy Morbidity in terms of major and minor complications was comparable among the three groups. Nonetheless, compared with the HAL-RP group, more patients from the TLRP group underwent reoperation. There were seven major complications in the TLRP group. Six patients were reoperated: five laparoscopically and one by open surgery because of necrosis of the ileostomy (Table 2). All patients who had a laparoscopic reintervention were given a protecting loop ileostomy except for one patient with a small-bowel herniation through the major omentum. All major complications requiring a reoperation in each of the three groups regarding anastomotic leak or abscess formation occurred in patients without a protecting ileostomy. Indications for reoperation in each of the three groups are provided in Table 2. A total of 16 patients who underwent TLRP had undergone a diversion procedure at the time of discharge from the hospital (12 primary, 4 secondary). In all these patients bowel continuity was restored within three months after surgery. In the HAL-RP group, 11 had undergone a diversion procedure at the time of discharge (8 primary, 3 secondary). In the ORP group, 11 patients were diverted (7 primary, 4 secondary). In ten patients from both groups, bowel continuity was restored within three months after surgery. In one patient from the HAL-RP group, bowel continuity was never restored because of the postoperative diagnosis of a cholangiocarcinoma. In one patient from the ORP group, bowel continuity could not be restored because of a persistent intra-abdominal abscess.9 There was a statistically significant shorter period to oral intake in the TLRP group compared with both other groups (time to resumption of liquid diet, 3 vs. 5 vs. 5 days and normal diet 5 vs. 6 vs. 7 days in the TLRP, HAL-RP, and ORP groups, respectively; P = 0.004 and P = 0.018, respectively). In spite of this, there was no statistical difference in hospital stay between the three groups (Table 2). Although daily and total morphine requirement in the TLRP was lower compared with the HAL-RP and ORP groups, this difference was not statistically significant (Fig. 1). Figure 1Daily and total morphine requirement after TLRP vs. HAL-RP vs. ORP. HAL-RP = hand-assisted laparoscopic restorative proctocolectomy; TLRP = total laparoscopic restorative proctocolectomy; ORP = open restorative proctocolectomy. Results of the SF-36 and GIQLI were comparable between the groups. A significant decline was found on all scales of the SF-36 (Fig. 2) and total GIQLI score (Fig. 3) in the first two weeks after the operation (P < 0.05). This decline was, however, not affected by the type of surgery (TLRP vs. HAL-RP vs. ORP; P > 0.05). QOL returned to baseline levels after four weeks and continued to improve until three months postoperatively, without any significant differences between the groups. Figure 2Results of postoperative recovery measured with SF-36 questionnaire (mean ± SEM). The x-axis represents the time when the questionnaires were completed, before and after surgery. The HAL-RP group is represented in green, the TLRP group is represented in grey, and the ORP group is represented in blue. HAL-RP = hand-assisted laparoscopic restorative proctocolectomy; TLRP = total laparoscopic restorative proctocolectomy; wks = weeks; mnths = months; SF-36 = Medical Outcomes Study Short Form 36. Figure 3Results of postoperative recovery measured with the GIQLI questionnaire (mean ± SEM). The x-axis represents the time when the questionnaires were completed, before and after surgery. The HAL-RP group is represented in green, the TLRP group is represented in grey, and the ORP group is represented in blue. HAL-RP = hand-assisted laparoscopic restorative proctocolectomy; TLRP = total laparoscopic restorative proctocolectomy; wks = weeks; mnths = months; GIQLI = Gastrointestinal Quality of life Index. A specification of costs is shown in Table 3. Costs for surgery were significantly higher in the TLRP group compared with both other groups, both because of the higher costs for material and the longer operating times. Total costs were 1864 euros less in the TLRP compared with the HAL-RP group. This difference, which was not statistically significant, can be explained by the shorter hospital stay in the TLRP group. Table 3Results of median costs after TLRP vs. HAL-RP vs. ORP (in Euros)  TLRPHAL-RPORPP value‡P value§P value∥Standardized costs Material during operation 2,849 2,347 1,071 — — — Use of OR, including personnel (Euro/min) 4.85 4.85 4.85 — — — Day of care at surgical ward 1,055 1,055 1,055 — — — Creating Ileostomy 87 87 87 — — — Closing Ileostomy 83 83 83 — — — Relaparotomy 87 87 87 — — — Relaparoscopy 169 169 — — — — Calculated costs for surgery (primary operation) Costs for personnel + OR use (adjusted for operating time) 1,445 (1,140–1,819) 1,040 (723–1,940) 645 (470–1,261) <0.001 <0.001 <0.001 Total costs of operation (personnel + OR use + material) 4,294 (3,989–4,668) 3,387 (3,070–4,287) 1,721 (1,541–2,332) <0.001 <0.001 <0.001 Calculated costs of admission Costs for primary hospital stay 9,495 (5,275–41,145) 10,550 (5,275–32,705) 11,605 (6,330–29,540) 0.139 — — Total costs for readmission for complications + stoma closure* 4,220 (1,055–31,650) 5,275 (5,275–16,880) 5,275 (4,220–22,155) 0.164 — — Costs for admission (all days of admission) 10,550 (5,275–69,630) 13,188 (5,275–42,200) 12,133 (7,385–43,255) 0.381 — — Total costs† 14,864 (9,312–75,384) 16,728 (8,364–46,468) 13,405 (9,145–45,466) 0.165 — — TLRP = total laparosopic restorative proctocolectomy; HAL-RP = hand-assisted laparoscopic restorative proctocolectomy; ORP = open restorative proctocolectomy; OR = operating room. Data are numbers or median costs per patient with ranges in parentheses unless otherwise indicated. *For readmitted patients only. †Total costs include all costs for surgery (use of OR + personnel, material, reoperations, costs for stoma), costs for admission, and costs for any readmission. ‡TLRP vs. HAL-RP vs. ORP (Kruskal-Wallis). §TLRP vs. HAL-RP (Mann-Whitney U test). ∥TLRP vs. ORP (Mann-Whitney U test). BODY.DISCUSSION: The present study demonstrated that compared with HAL-RP and ORP, TLRP offers no clinically significant short-term advantages, apart from an earlier return to diet. Moreover, it is associated with longer operating times and a tendency to a higher reintervention rate. It can be concluded that costs are comparable as the reduction in overall costs after TLRP counterbalances the increased surgical costs. The presumption that a total laparoscopic approach in restorative proctocolectomy is associated with more favorable short-term results compared with a hybrid HAL-RP procedure could not be substantiated. Critics might argue that the applied total laparoscopic approach is a laparoscopic-assisted rather than a pure laparoscopi7 Consequently, the pouch is stapled laparoscopically, and a handsewn ileoanal anastomosis is performed transperineally after mucosectomy. In the latter procedure, the ileoanal anastomosis is performed extracorporeally, whereas in our approach the anastomosis is made laparoscopically. Although not significantly so, the rate of reintervention was doubled after TLRP compared with HAL-RP but not because of anastomotic leak or pelvic abscess. Proportionally more patients in the TLRP group had UC (86 vs. 67 vs. 67 percent in the TLRP, HAL-RP, and ORP groups, respectively). Consequently, these patients were exposed to higher doses of immunosuppressive drugs. This also explains why more patients from the TLRP group received a primary protecting loop ileostomy (Table 1). Within all three groups, reoperations only occurred in those patients without a protecting ileostomy, except for one patient in the TLRP group who underwent reoperation for stoma necrosis. Kienle et al.1 is one of the few authors who applied a total laparoscopic approach as well. In a consecutive series of 59 total laparoscopic proctocolectomies, the authors reported comparable operating times but an increased complication rate and an increased length of hospital stay compared with the present study. Moreover, conversion rate in the study by Kienle et al.1 was considerable (8 percent). The total laparoscopic approach, comprising a right hemicolectomy, left hemicolectomy, and rectum excision, probably makes the operation more vulnerable to complications, because the procedure is more difficult compared with, for instance, HAL-RP or open surgery. Several studies comparing hand-assisted and true laparoscopic surgery for other procedures, such as sigmoidectomy or left hemicolectomy, concluded that the hand-assisted laparoscopic approach reduced operating times without affecting recovery.8–12 Little evidence is available regarding the role of hand-assisted laparoscopic surgery for restorative proctocolectomy. A study by Nakajima et al.,10 including only a small number of patients, noticed an increase in operating time using a pure laparoscopic compared with a hand-assisted technique, with equal morbidity and hospital stay. Noticeably, Larson et al.13 observed an increase in operating time after HAL-RP compared with TLRP. Hospital stay was one day shorter in the TLRP group, and in 6 percent of the cases a conversion to open surgery was necessary. In the present study, a stoma was constructed in only one-third of the patients and no conversions occurred. Because patients with a protecting loop ileostomy are known to have a shorter primary hospital stay, the present study and that of Larson et al.,13 in which all patients received a covering loop ileostomy, cannot readily be compared with respect to length of hospital stay. So what is there to gain from a TLRP compared to a HAL-RP? In the present study, a significantly quicker extend to full liquid and normal diet was observed, which can partially be explained by the fact that almost half of the patients from the TLRP compared with one-third of patients in the HAL-RP and ORP groups received a protecting ileostomy. Obviously, this is of limited clinical relevance because it did not significantly influence length of hospital stay. However, the pouch catheter, residing for six days according to protocol, might have tempered the potential benefits of an earlier extend to normal diet in those patients without a protecting ileostomy. Interestingly, a reduction in hospital stay of two days after TLRP compared with ORP was observed as opposed to a difference of one day compared with HAL-RP. This decrease was not statistically significant. There are some limitations to this study that could explain some of the differences in outcome between the study groups. As stated above, more patients in the TLRP group received an ileostomy. Because oral intake in patients with a defunctioning ileostomy is generally advanced quicker, this might have influenced the results. Another form of bias might be the fact that the more recently obtained results after TLRP were compared with a historic cohort of patients. The implementation of a fast-track care program for segmental colectomies in August 2004 at one of our surgical wards might have precipitated advancement of oral intake and mobilization. In this way, although discharge criteria and perioperative treatment in patients who underwent TLRP were similar to those who underwent HAL-RP and ORP between 2000 and 2003, some of the discharge criteria might have been fulfilled earlier. Finally, an allocation bias for laparoscopic surgery has been a possible confounding factor, because all patients referred to the laparoscopic surgeon were operated on by using a total laparoscopic approach, except for two patients who favored an open approach. However, compared with the HAL-RP, patients from the TLRP had to some extent less favorable preoperative characteristics, such as an older age, higher body mass index, and more patients with ulcerative colitis. Cosmesis after laparoscopic restorative proctocolectomy is considered superior to that after ORP. Although cosmesis after TLRP might be superior to that after HAL-RP, it is difficult to believe that a reduction of 2 cm in the size of the Pfannenstiel incision might influence cosmesis and body image significantly.14 There are no clear short-term advantages after TLRP compared with HAL-RP with respect to recovery, quality of life, and costs. There is however one important argument for TLRP: it is an excellent procedure for training surgeons in laparoscopic colorectal surgery because it comprises a total left and right hemicolectomy and a laparoscopic proctectomy. In teaching hospitals, this might compensate for the prolonged operating times. BODY.CONCLUSIONS: A total laparoscopic technique for restorative proctocolectomy, apart from an earlier extend to normal diet, has no clinically relevant short-term advantages compared with an open and hand-assisted laparoscopic technique, because operating times are further prolonged, hospital stay and QOL are similar, and costs are comparable.

TITLE: The NSIGHT1-randomized controlled trial: rapid whole-genome sequencing for accelerated etiologic diagnosis in critically ill infants Genetic disorders are a leading cause of morbidity and mortality in infants in neonatal and pediatric intensive care units (NICU/PICU). While genomic sequencing is useful for genetic disease diagnosis, results are usually reported too late to guide inpatient management. We performed an investigator-initiated, partially blinded, pragmatic, randomized, controlled trial to test the hypothesis that rapid whole-genome sequencing (rWGS) increased the proportion of NICU/PICU infants receiving a genetic diagnosis within 28 days. The participants were families with infants aged <4 months in a regional NICU and PICU, with illnesses of unknown etiology. The intervention was trio rWGS. Enrollment from October 2014 to June 2016, and follow-up until November 2016. Of all, 26 female infants, 37 male infants, and 2 infants of undetermined sex were randomized to receive rWGS plus standard genetic tests (n = 32, cases) or standard genetic tests alone (n = 33, controls). The study was terminated early due to loss of equipoise: 73% (24) controls received genomic sequencing as standard tests, and 15% (five) controls underwent compassionate cross-over to receive rWGS. Nevertheless, intention to treat analysis showed the rate of genetic diagnosis within 28 days of enrollment (the primary end-point) to be higher in cases (31%, 10 of 32) than controls (3%, 1 of 33; difference, 28% [95% CI, 10–46%]; p = 0.003). Among infants enrolled in the first 25 days of life, the rate of neonatal diagnosis was higher in cases (32%, 7 of 22) than controls (0%, 0 of 23; difference, 32% [95% CI, 11–53%];p = 0.004). Median age at diagnosis (25 days [range 14–90] in cases vs. 130 days [range 37–451] in controls) and median time to diagnosis (13 days [range 1–84] in cases, vs. 107 days [range 21–429] in controls) were significantly less in cases than controls (p = 0.04). In conclusion, rWGS increased the proportion of NICU/PICU infants who received timely diagnoses of genetic diseases. ABSTRACT.WHOLE GENOME SEQUENCING: SPEEDIER DIAGNOSES IN INFANTS: Genetic disorders in critically ill infants can be diagnosed in as little as 26 h by rapid whole genome sequencing (rWGS). A study led by Stephen F. Kingsmore at the Rady Children’s Institute for Genomic Medicine in San Diego and Children’s Mercy Hospital in Kansas City compared the time to genetic diagnosis in 65 infants with inherited diseases of unknown cause using rWGS, clinical confirmatory testing and standard genetic tests or standard genetic tests alone. They found that the addition of rWGS including confirmatory testing significantly decreased the time to diagnosis, which in newborns can mean the difference between life and death. Because of the increasing accessibility and decreasing costs of the technology and the critical need for timely and effective intervention in infants with suspected genetic diseases, the authors advocate the use of rWGS as a first-line diagnostic test. ABSTRACT.INTRODUCTION: A premise of pediatric precision medicine is that outcomes are improved by replacement of clinical diagnosis and empiric management with genetic diagnosis and genotype-differentiated treatment.1–9 The evidence base for pediatric precision medicine is still underdeveloped.10, 11 Ill infants are especially in need of precision medicine since genetic diseases are a leading cause of mortality, particularly in neonatal intensive care units (NICU) and pediatric intensive care units (PICU).5–7, 12–16 Among high-cost health care, NICU treatment is one of the most cost-effective.17–19 Since disease progression can be very rapid in infants, genetic diagnoses must be made quickly to permit consideration of precision interventions in time to decrease morbidity and mortality.5,6, 20–23 For a few genetic diseases, newborn screening has shown early neonatal diagnosis and rapid, precise intervention to dramatically improve outcomes.24, 25 The potential expansion to newborn diagnosis for symptomatic infants for all 5000 genetic diseases26 has been made technically possible by the advent of clinical genomic sequencing (whole-genome sequencing (WGS) or whole-exome sequencing (WES), and next-generation sequencing gene panel tests (NGS). In particular, rapid WGS (rWGS) can allow genetic diagnosis in 2 days.20, 27 There is substantial evidence that a higher proportion of symptomatic children with likely genetic disease receive etiologic diagnoses by WGS and WES than other genetic tests.3–7, 28–35 Published NICU or PICU experience with rWGS, however, is limited to case reports and one retrospective study.5,6, 20–23 In the latter, 57% of infants received genetic diagnoses in a median of 23 days (day of life 49).6 However, it has not yet been unequivocally demonstrated whether rWGS improves timeliness of genetic diagnosis relative to standard genetic tests. Here we report results of newborn sequencing in genomic medicine and public health randomized controlled trial (RCT) 1 (NSIGHT1), an RCT of genomic testing in patients (ClinicalTrials.gov Identifier: NCT02225522).24 Specifically, NSIGHT1 compared rates of genetic diagnosis in NICU and PICU infants with possible genetic diseases at 28 days from enrollment by standard tests alone vs. standard tests plus trio rWGS. ABSTRACT.RESULTS: ABSTRACT.PATIENTS: Of 129 nominated infants, 65 (50%) completed the NSIGTH1 study (Figs. 1 and 2). Sixty four enrollees were NICU infants. The infants nominated represented 7% of NICU and PICU admissions during this interval. Thirty-two infants randomized to rWGS plus standard genetic tests (cases) and 33 to standard tests alone (controls, Figs. 1 and 2). The baseline characteristics of the infants were similar in the two arms and similar to those of a previous retrospective case series of infants receiving rWGS in this NICU and PICU (Table 1).6 Detailed (deep) phenotypes of infants were extracted from the electronic medical record in 42 infants receiving genomic sequencing, since this was a prerequisite for interpretation. On average, infants receiving rWGS had 5.9 phenotypic features (range 1–17; Table S1). Phenotypes were highly diverse and typically present at birth (Table 1, S1). The most common indications for nomination were congenital anomalies (35%) and neurological disorders (25%; Table 1). Fewer control infants had cardiovascular findings (6 vs. 28%; difference, −22% [95% CI, −40 to −4%]; p = 0.02) than cases, which may have affected likelihood for genetic disease (Table 1).Fig. 1Design of “Newborn Sequencing In Genomic medicine and public HealTh” study 1 (NSIGHT1; ClinicalTrials.gov accession NCT02225522). Time (t) is in days. WGS whole-genome sequencing, EHR Electronic Health Record Fig. 2CONSORT flow diagram of NSIGHT1 enrollment and randomization. Major reasons for non-enrollment were family refusal (13%), the infant had a diagnosis that explained the phenotype (9%), and incomplete nominations (9%). At unblinding of clinicians (by 10 days after enrollment), a provision was made whereby clinicians could request compassionate cross-over to the rWGS group if the infant was critically ill. Cross-over was requested for 7 (21%) of 33 infants who randomized to standard tests alone, of which 5 met these criteria and were granted Table 1Characteristics of the 65 NSIGHT1 probands Cases (rWGS, n = 32)Controls (n = 33)Sex Female (n, %) 15 (47%) 11 (33%) Male (n, %) 16 (50%) 21 (64%) Undetermined (n, %) 1 (3%) 1 (3%) Demographics Caucasian (n, %) 25 (78%) 27 (82%) African, African American (n, %) 2 (6%) 1 (3%) Other race (n, %) 5 (16%) 5 (15%) Hispanic (n, %) 2 (6%) 3 (9%) Consanguinity (n, %) 1 (3%) 2 (6%) Birth characteristics Gestational age (average, wks) 36.0 35.9 Weight (average, kg) 2.5 2.4 Low birth weight (<2500 g, n, %) 14 (44%) 9 (27%) Extremely low birth weight (<1000 g, n, %) 1 (3%) 3 (9%) APGAR at 1 min (average) 6.1 5.1 APGAR at 5 min (average) 7.8 6.4 Symptom Onset (average day of life) 2.3 2.1 Primary system involved by disease Congenital anomalies/musculoskeletal 10 (31%) 13 (39%) Neurological 5 (16%) 11 (33%) Cardiovascular findings 9 (28%) 2 (6%) Endocrine/metabolic 1 (3%) 3 (9%) Respiratory findings 4 (13%) 0 (0%) Renal 1 (3%) 2 (6%) Dermatologic 1 (3%) 0 (0%) Multiple system 1 (3%) 0 (0%) Hepatic 0 (0%) 0 (0%) Enrollment and standard clinical testsa Day of life at enrollment (average, range) 22.8 (1–101) 22.0 (1–80) Probands receiving standard clinical tests (n, %) 30 (93.8%) 33 (100%) Day of life 1st standard clinical test ordered (average, range) 11.6 (0–66) 15.6 (0–120) All standard clinical tests ordered (average, range) 2.8 (0–7) 3.4 (1–10) Probands receiving Standard Clinical NGS panels, WES or WGS (n, %) 17 (53%) 24 (73%) Standard Clinical NGS panels, WES and WGS Tests Ordered (n, range) 22 (0–2) 43 (0–4) Probands receiving rWGSb 32 (100%) 5 (15%) Genetic disease diagnoses Diagnosis (Standard Clinical Test or rWGS; n, %)b 13 (41%) 8 (24%) Diagnosis by Standard Clinical Tests (n, %) 7 (22%) 8 (24%) Diagnosis by rWGS (n, %)b 10 (31%) 2 (6%) DOL diagnosis by Standard Clinical Test (median, range) 66 (16–151) 130 (37–451) Time to Diagnosis by Standard Clinical Test (average, range) 45 (16–150) 110 (31–450) aThe statistics for standard clinical tests exclude newborn screening, which all infants received, and did not result in any diagnoses bIncludes Controls 5007, 5012, 5029, 5040 and 5053, which were crossed over to rWGS ABSTRACT.STANDARD DIAGNOSTIC TESTS: Standard diagnostic tests for genetic diseases were performed as clinically indicated in 63 of the 65 infants (Table 1). They included all postnatal diagnostic tests that could be ordered through the electronic medical record. The proportion of infants receiving standard genetic tests and age at first standard test order were similar in both arms (Table 1). In addition to newborn screening infants received an average of 3.1 (range 0–10) standard genetic tests (Table 1, S3), which was similar to a previous retrospective case series of infants receiving rWGS from the same NICU and PICU.6 During the study, non-expedited WGS became available as a standard diagnostic test. Of 33 control infants, 24 (73%) received non-expedited clinical NGS panel tests, WES or WGS standard tests, compared with seventeen (53%) of 32 cases (Table 1, S3). Other than newborn screening, the average age at first standard test order was 14 days (range 0–120 days). Standard tests yielded fifteen (24%) genetic diagnoses in the 63 subjects tested, seven (23%) in 30 cases, and eight (24%) in 33 controls (Table 2, S4). The rates of diagnosis by individual standard clinical tests were: chromosomal microarray 6% (three of 48 tests); Clinical NGS panel test 18% (nine of 49 tests); Clinical WES 33% (one of three tests); Methylation 13% (one of eight). Of note, five (33%) of 15 diagnoses by standard tests would not have been detected by rWGS at the time of study: four were copy number or structural variants and one was a change in DNA methylation. The median time from first standard test order to diagnosis was 64 days (range 16–450 days). The average age at diagnosis by standard genetic tests was 113 days (range 16–451 days). Six (10%) of 63 infants received a diagnosis by standard tests prior to hospital discharge (Table S5).Table 2Presentations and characteristics of the twenty one infants who received diagnoses (Dx) of genetic diseases Patient IDStudy armDx typeMode of DxPrimary clinical featuresaDiagnosisGeneInheritance patternDe novo or inheritedVariant chromosomal (Chr)b or gene (c.) coordinateVariant patho-genicityVariant protein coordinate5004 Case Partial Std Cleft palate micrognanthia hypoglycemia hyperinsulinimia thrombocytopenia Chr 7p duplication syndrome n.a. n.d. n.d. Gain 7p22.3-p15.2 Chr7:43360-26463160dup P n.a. 5007 Control Full rWGS and Std Polymicrogyria intractable seizures epileptic encephalopathy Congenital disorder of glycosylation type Ik ALG1 Autosomal Recessive Inherited c.15 C > A and c.149 A > G P and LP p.C5* and p.Q50R 5008 Case Full Std Complete atrioventricular canal defect hypospadias IUGR dysmorphic features Chr 8p23 deletion syndrome n.a. n.d. n.d. Chr8:158048-6999114del 10054927-10479436dup 10479473-11882401del P n.a. 5011 Control Full Std Hypotonia cryptorchidism aniridia XL myotubular myopathy-1 MTM1 X-Linked Recessive; n.d. c.137-3 T > G; P n.a. Aniridia PAX6 Autosomal Dominant Inherited c.1268 A > T P p.*423 L 5014 Control Full Std Hyperglycemia Transient neonatal diabetes ZFP57 n.d. n.a. Hypomethylation 6q24 P n.a. 5023 Case Full rWGS and Std Hyponatremia SGA/IUGR pseudohypoaldosteronism Pseudohypoaldosteronism type I NR3C2 Autosomal Dominant Inherited c.1951C > T LP p.R651* 5025 Control Full Std Micrognathia cleft palate abnormal facies right thumb hypoplasia Nager type acrofacial dysostosis SF3B4 Autosomal Dominant de novo c.1088-3 C > G LP n.a. 5026 Control Full Std Hirsutismmild synophrys mild micrognathia camptodactyly renal cysts Cornelia de lange syndrome 1 NIPBL Autosomal Dominant de novo c.5057del P p.L1686Rfs*7 5027 Control Full Std IUGR cleft palate Micrognathia Skin tags Poor gag reflex Chr 1p36 deletion syndrome n.a. Autosomal dominant de novo Loss arr 1p36.11 Chr1:24100645-25003678del LP n.a. 5030 Case Full Std Seizures poor feeding AD nocturnal frontal lobe epilepsy CHRNA4 Autosomal Dominant de novo Heterozygous deletion of CHRNA4 LP n.a. 5035 Case Full rWGS Microcephaly Primary AR microcephaly 5 ASPM Autosomal Recessive Inherited c.3428dupT; c.8191_8192del P,P p.L1144Vfs*16; p.E2731Kfs*19 5036 Case Full rWGS Central apnea Congenital central hypoventilation syndrome PHOX2B Autosomal Dominant de novo PHOX2B ALA EXP P p.A260(9) 5038 Case Full rWGS Situs inversus Primary ciliary dyskinesia type 7 DNAH11 Autosomal Recessive Inherited c.6244 C > T; c.6776 A > T and c.8567 T > C P, LP, LP p.R2082*; p.D2259V and p.V2856A 5042 Case Full rWGS Profound hypotonia Respiratory distress Myoclonic jerks AD mental retardation 31 PURA Autosomal Dominant de novo c.458_459dupC P p.K154Qfs*47 5048 Case Full rWGS Seizures Early infantile epileptic encephalopathy 14 KCNT1 Autosomal Dominant de novo c.1420 C > T P p.R474C 5051 Case Full rWGS Perinatal ascites; cholestasis Dehydrated hereditary stomatocytosis PIEZO1 Autosomal Dominant Inherited c.6058 G > A P p.A2020T 5053 Control Full rWGS and Std Altered mental status Decreased deep reflexes Hypotonia cryptorchidism XL myotubular myopathy MTM1 X-linked Recessive de novo c.567_569delTAA P p.N189del 5057 Case Full rWGS and Std Dysmorphic features Cardiac anomalies failed hearing screen Noonan syndrome SOS1 Autosomal Dominant de novo c.2536 G > A P p.E846K 5059 Case Full rWGS and Std HLHS hydrocephalus multiple congenital anomalies Coffin–siris syndrome ARID1A Autosomal Dominant de novo c.1207 C > T LP p.Q403* 5061 Case Partial rWGS and Std Hypotonia absent gag reflex exaggerated startle reflex Hyperekplexia GLRA1 Autosomal Dominant de novo c.373 G > A LP p.D125N 5062 Control Full Std Bicuspid aortic valve, hypotonia, leukocytosis Central core disease of muscle RYR1 Autosomal Dominant de novo c.14581 C > T P p.R4861C Controls 5007, 5012, 5029, 5040 and 5053 were crossed over to rWGS Chr chromosome, std standard genetic test, P pathogenic, LPlikely pathogenic, n.d. not determined *Premature stop codon created aFull clinical features are shown in Table S1 bGRCh37 ABSTRACT.RAPID WHOLE-GENOME SEQUENCING: rWGS was performed on infant-parent trios with Illumina HiSeq instruments, with paired reads to an average depth of 40-fold, detecting an average of 5.0 million nucleotide variants per genome (standard deviation 0.3 million variants; Table S2, Figure S1). Ten of 32 cases (31%) received diagnoses by rWGS (Table 2, Table S4). Upon un-blinding of clinicians to randomization at day 10 after enrollment, compassionate cross-over to rWGS was requested for seven (21%) of the 33 controls. Cross-over to rWGS was declined in two infants who were not acutely ill; both were about to be discharged to home, with follow-up of their medical conditions as outpatients. Five cross-over requests were granted, yielding two diagnoses. In both, diagnosis by rWGS occurred first but was recapitulated by standard tests (Table 2). Including five crossovers, 12 (32%) of 37 infants received rWGS diagnoses (Table 2, S5). On average, enrollment occurred on DOL 22 (range 1–101; Table 1), which was earlier than in our previous report of rWGS (DOL 26; Table 1),6 but an average of 8 days later than standard tests. The median time to rWGS diagnosis, including clinical confirmatory testing, was 14 days (range 8–35 days; Table S5), which was also faster than our previous report of rWGS (23 days; Table S5).6 The median age at diagnosis in patients randomized to rWGS was 28.5 days (range 14–90 days). Among crossovers, the median age at rWGS diagnosis was 94.5 days. The research protocol required confirmation of rWGS results by another method prior to clinical reporting except in cases where life-threatening progression was imminently likely. There were no such cases, and no provisional diagnostic reports of rWGS results were returned prior to confirmatory testing. Sanger sequencing confirmed all rWGS results. ABSTRACT.DIAGNOSES: Twenty-two genetic diagnoses were reported in 21 (32%) of 65 infants (Table 2). Thirteen cases (41%) received diagnoses by rWGS or standard tests. Eight controls (24%) received diagnoses (Table 1). One individual received two diagnoses. Only one diagnosis was recurrent (X-linked myotubular myopathy in two infants), reflecting substantial genetic heterogeneity among NICU disease presentations6 (Table 2). The most common mechanism was de novo variant occurrence (12 of 21 (57%) diagnoses; Table 2). Seventeen (65%) causative variants were reported as pathogenic, and nine (35%) as likely pathogenic. Eight variants (31%) were predicted to result in amino acid substitutions, six (23%) were indels, five (19%) were predicted to result in stop codon loss or gain, four (15%) were structural variations, two (8%) were predicted to alter splicing, and one (4%) impacted methylation. The most common inheritance pattern was autosomal dominant (14 of 19 (74%) diagnoses), followed by autosomal recessive (three, 16%) and X-linked recessive (two, 11%). In 19 of 21 (90%) diagnosed probands, the diagnosis explained all of the clinical features (Table 2). Two possible diagnoses provoked discussion regarding inclusion. Control infant 5053 had altered mental status, decreased deep tendon reflexes, hypotonia, and cryptorchidism (Table S1). He had a duplication of Chr 1p36.32 as well as X-linked Myotubular Myopathy. Chr 1p36.32 duplication syndrome was not included as a diagnosis. The second, paternally inherited SCN5A c.6010 T > C (p.F2004L) in Case infant 5033 with persistent, symptomatic atrial fibrillation/flutter (Table S1), was not included as a diagnosis. While several case studies had reported this variant as pathogenic and two functional studies showed it to be deleterious, the allele frequency (0.3%) was considered too high to be likely pathogenic, and a diagnosis of familial atrial fibrillation type 10 was not reported.36–40 ABSTRACT.CLINICAL UTILITY OF MOLECULAR DIAGNOSES: The short-term clinical impact of diagnoses was assessed by chart reviews and surveys with referring physicians (Table S4). Thus, clinical utility reflected actual practice, rather than an ideal or maximal personalization of treatment. Clinical utility did not include the impact of negative test results on management. 20 (31%) of the 65 infants enrolled (95% of those receiving diagnoses) had a consequent change in management. Ten (15%) infants (48% of those receiving diagnoses) had a change in management other than genetic or reproductive counseling of parents. Diagnoses were not associated with any harms. ABSTRACT.EARLY STUDY TERMINATION: The study was terminated after 21 months due to growing availability of targeted NGS panels, WES and WGS as standard tests, which shifted the baseline of comparison over the course of the study. These were associated with high rates of cross-over requests and higher utilization of targeted NGS panels, WES or WGS as standard clinical genetic tests among controls (73%) than cases (53%; Table S3). ABSTRACT.END-POINT TESTING: End-points were analyzed on the basis of intention to treat (Figs. 1 and 2). Thus all patients were analyzed in the groups to which they were randomized. The primary end point, rate of genetic diagnosis within 28 days of enrollment, was higher in cases (31%, ten of 32) than controls (3%, one of 33; difference, 28% [95% CI, 10–46%]; p = 0.003 Table 3). Kaplan–Meier curves supported the conclusion that there was a significantly higher probability of receiving a diagnosis by rWGS until DOL 99 or 67 days after test order (Fig. 3). For neonates enrolled within the first 25 days of life, the rate of diagnosis by DOL 28, a secondary end-point, was higher in cases (32%, seven of 22) than controls (0%, zero of 23; difference, 32% [95% CI, 11–53%]; p < 0.01; Table 3). In practice, crossovers did not materially affect these end-points, since the two diagnoses made by rWGS among five cross-over infants occurred later than DOL 28 and 28 days after enrollment (Table S3).Table 3Comparison of primary and secondary end-points rWGS + standard testingStandard testing (including crossovers)P-valueStatistical testNumber of subjects 32 33 Primary end-point Diagnosis within 28 days of enrollment (n, %) 10 (31%) 1 (3%) 0.003a Fisher’s exact test Secondary end-points Diagnosis by DOL 28 (n, %) 7 (32%) 0 (0%) 0.004a Fisher’s exact test Total diagnoses (n, %) 13 (41%) 8 (24%) 0.19 Fisher’s exact test Clinical utility of diagnoses (n, %) 13 (41%) 7 (21%) 0.11 Fisher’s exact test DOL hospital discharge (average, range) 66.3 (3–456) 68.5 (4–341) 0.91 Two sample t-test Diagnosis before discharge (n, %) 9 (28%) 3 (9%) 0.06 Fisher’s exact test  Mortality at 180 days (n, %) 4 (13%) 4 (12%) n.d. Age at death (days; median, range) 62 (14–228) 173 (4–341) 0.93 Log rank test aFisher’s exact test p-value both for all patients and in a sensitivity analysis, in which patients with a partial diagnosis (5004 and 5061) where considered undiagnosed DOL day of life Fig. 3Kaplan–Meier curves of time to diagnosis in cases and controls. The cumulative probability of a diagnosis (Dx) in cases (infants randomized to receive rWGS plus standard genetic tests; shown in red; n = 32) and controls (infants randomized to standard genetic tests alone; shown in blue; n = 33). Differences in probability of receiving a diagnosis were significant between the two arms from day 12–67 after enrollment (a asterisks) and DOL 19 - 99 (b asterisks) Age at diagnosis and time to diagnosis differed significantly between arms, after accounting for non-proportional rates of diagnosis (Table 4, Table S5): The median age at diagnosis in cases was 25 days (range 14–90 days) vs. median in controls was 130 days (range 37–451). The median time to diagnosis in cases was 13 days (range 1–84 days) vs. median in controls 107 days (range 21–429 days). Two diagnoses explained part of the infant’s disorder rather than the entire presentation. In a sensitivity analysis, when patients adjudged to have a partial diagnosis were considered undiagnosed, age at diagnosis and time to diagnosis were no longer significant (Table 4).Table 4Comparison of age at diagnosis and time to diagnosis between cases (rWGS plus standard tests) and controls (standard tests alone) Original analysisaSensitivity analysisbp-value for non-proportional hazardsp-value for a difference in overall diagnostic ratesp-value for non-proportional hazardsp-value for a difference in overall diagnostic ratesAge at diagnosis 0.002 0.043 0.003 0.15 Time to diagnosis from enrollment 0.002 0.040 0.002 0.11 aPeto–Peto test used instead of log-rank test due to evidence of non-proportional hazards bPeto–Peto test when patients with a partial diagnosis (5004 and 5061) considered undiagnosed. Six other secondary end-points did not differ significantly between arms in an intention to treat analysis (Tables 1, 3, 4, S4). They were the proportion of infants receiving diagnoses of genetic diseases (41% of cases vs. 24% of controls; difference, 16% [95% CI, −6 to 39%])), proportion in whom diagnoses had clinical utility (41% of cases vs. 21% of controls; difference, 19% [95% CI, −3 to 42%]), proportion of infants with a change in medical management (clinical utility, 22% of cases vs. 9% of controls; difference, 13% [95% CI, −5 to 30%]), proportion of patients who received diagnoses prior to hospital discharge (28% of cases vs. 9% of controls; difference, 19% [95% CI, 0–38%]), average length of NICU/PICU stay (average 67 days), 6-month mortality (12%, 8 of 65), and age at death. ABSTRACT.DISCUSSION: NICU and PICU infants receiving trio rWGS plus standard clinical testing had a higher rate of genetic diagnosis and shorter time to diagnosis than infants receiving standard tests alone. In intention to treat analysis, rWGS was associated with significantly more genetic diagnoses within 28 days of enrollment (31%, 10 of 32) than standard tests alone (3%, 1 of 33; difference, 28% [95% CI, 10–46%]; p = 0.003). The rate of neonatal (DOL 28) diagnosis was higher in cases (32%, 7 of 22) than controls (0%, 0 of 23; difference, 32% [95% CI, 11–53%]; p = 0.004). Of note, standard genetic testing was ordered an average of 8 days before enrollment, which benefitted the control arm over rWGS cases for these analyses. Nevertheless, age at diagnosis and time to diagnosis were significantly shorter in rWGS cases, after accounting for non-proportional rates of diagnosis. The rate of genetic diagnosis by rWGS in a NICU or PICU was reported previously in one cohort.6 Enrollment in that study was at average DOL 26 (vs. DOL 22 herein). The rate of diagnosis by rWGS therein was 14% (5 of 35) by DOL 28, and 34% (12 of 35) within 28 days of enrollment, which were similar to herein (32% and 31%, respectively). The total rate of genetic diagnosis by rWGS herein (32%) was within the range reported for WGS and WES studies.3–7, 28–35 Timely return of rWGS diagnoses was limited by two research factors that may not be part of routine clinical practice: firstly, confirmatory testing by “the clinically accepted standard” was required for research rWGS diagnoses—but was not necessarily required for laboratory developed, clinical WGS, WES, and targeted NGS panel tests—which lengthened the time to rWGS diagnosis by 7–10 days. Indeed, all diagnostic rWGS findings in the current study were concordant with orthologous methods. For well covered, pathogenic and likely pathogenic, single nucleotide variants in regions of high WGS quality, a median time-to-result of 5 days is anticipated.6,20, 27 Secondly, enrollment occurred relatively late during the NICU or PICU stay (DOL 22). While parents are interested in receipt of genomic sequencing at birth, an enrollment rate of 6% was reported for WES in NICU infants in another cohort.41, 42 Delay in enrollment herein reflected two logistical factors. First, since a criterion for enrollment was suspicion by the provider of an underlying genetic disease, nomination was often delayed until a genetic test or consult had been ordered. In such cases, the time of enrollment delayed the study test, rWGS, compared to standard testing; nevertheless, there was still a decreased time to diagnosis with rWGS. Secondly, NSIGHT1 required informed consent from both parents; the logistics and complexity of obtaining informed consent in a NICU or PICU setting are arduous. A follow-on study, NSIGHT2, has started in which enrollment occurs close to the day of NICU or PICU admission (ClinicalTrials.gov Identifier: NCT03211039). This was facilitated by simpler enrollment criteria, requirement of informed consent from a single parent, and limiting eligibility for enrollment to within several days of admission. Since the current study, clinical rWGS has improved with respect to rate of genetic diagnosis and time to diagnosis.27 In particular, the diagnostic rate has increased through ongoing identification of novel disease genes, improved reference genome sequences, and better identification of disease-causing copy number, repeat expansion, regulatory, splicing and structural variations.32, 43–50 These recent advances were not reflected in the current study. Indeed, in three cases herein, causative chromosomal deletions were detected by microarray but not by rWGS. rWGS has recently also become much more feasible in clinical laboratories due to improved throughput of rapid sequencing instruments (Illumina NovaSeq 6000), and the availability of robust commercial interpretation software. Rapid trio exome sequencing (rWES) has also become feasible, and has demonstrated similar performance to rWGS51: In a recent study, rWES revealed a molecular diagnosis in 51% of infants at an average of 33 days of life, and with a mean turnaround time of 13.0 days.51 Randomized, controlled studies are needed that compare the diagnostic and clinical utility and cost effectiveness of rWES and rWGS in NICU and PICU infants NSIGHT1 was terminated early, primarily due to loss of equipoise noted by some nominating clinicians during the study. Some practitioners grew to regard randomization to standard tests alone to be an inferior intervention than standard tests plus trio rWGS. This was associated with seven (21% of controls) requests to cross-over control infants to the rWGS arm following clinician un-blinding, five of which were granted. It was also associated with a higher rate of order of targeted NGS panels, WES or WGS standard genetic tests in controls (43 tests in 24 controls) than cases (22 tests in 17 cases). Standard genomic sequencing tests accounted for 63% (5) of the eight genetic diagnoses in controls. As a result, there was not a significant difference between arms in the total number of genetic diagnoses, a secondary end-point (41% [13] diagnoses among 32 infants in the rWGS arm, 24% (8) of 33 in controls; difference, 16% [95% CI, −6 to 39%]; p = 0.19). Future pragmatic RCT designs in genomic medicine will require careful attention to the principle of equipoise and to the rapid evolution of clinical NGS-based testing.52, 53 The more widespread use of gene panel testing in the NICU during the course of this study was a significant departure from our experience at study conception. Our study was not intended to evaluate the relative diagnostic yield of panel testing over rWGS. Consequently, the study was not powered to evaluate the non-inferiority of panels over rWGS. The rationale for rWGS in NICU infants is to enable consideration of acute precision interventions in time to decrease morbidity and mortality.5,6, 21–24 In two prior studies of genomic sequencing in infants, genetic diagnoses led to precision medicine that was considered life-saving in 5%, and that avoided major morbidity in 6%.6, 7 In those studies, early diagnosis (DOL 49) led to greater implementation of precision medicine (65%) than later diagnosis (DOL 374, 39%), particularly with regard to palliative care guidance. As in the current study, assessments of clinical utility were based on actual changes in management, which were limited by clinician experience with genomic medicine and rare genetic diseases. This is a major challenge for NICU and PICU implementation of genomic medicine for rare genetic diseases.54, 55 Unfortunately, early termination of the current study resulted in loss in power for the secondary end-points: there were not significant differences in the overall rate of clinical utility of diagnoses, length of admission, rate of diagnosis before discharge, mortality and age at death. The clinical utility of diagnoses and rate of diagnosis before hospital discharge trended towards being higher in the rWGS arm (difference, 19% [95% CI, −3 to 42%], p = 0.11, and 19% [95% CI, 0–38%], p = 0.06, respectively). Additional studies are needed to clarify whether shorter time to diagnosis is associated with changes in clinical utility of diagnoses, outcomes, or healthcare utilization. ABSTRACT.CONCLUSIONS: Among infants with suspected genetic diseases in a regional NICU or PICU, the addition of rWGS decreased the time to diagnosis. Since genetic diseases are among the leading cause of death in the NICU and PICU, as well as overall infant mortality, implementation of rWGS is likely to have broad implications for the practice of neonatology. ABSTRACT.METHODS: ABSTRACT.TRIAL DESIGN: NSIGHT1 tested the a priori hypothesis that rWGS increases the proportion of infants receiving a genetic diagnosis within 28 days in a partially blinded, randomized controlled study in a regional NICU and PICU in a tertiary referral children’s hospital (Children’s Mercy—Kansas City, CM-KC)(Fig. 1). Infants were born at CM-KC or transferred from outside birthing hospitals to CM-KC for intensive care at various ages. Inclusion criteria were infants in the NICU or PICU of age less than four months with illnesses of unknown etiology and one of the following: 1. A genetic test order or genetic consult; 2. A major structural congenital anomaly or at least three minor anomalies; 3. An abnormal laboratory test suggested a genetic disease; or 4. An abnormal response to standard therapy for a major underlying condition. Exclusion criteria were a previously confirmed genetic diagnosis that explained the clinical condition, or features pathognomonic for a chromosomal aberration. The NICU census was reviewed daily for eligible infants by enrollment coordinators. The eligibility criteria did not change after trial commencement. NICU clinicians were notified of eligible infants, who were nominated through a standard form. NICU and PICU clinicians notified families of eligible infants about the study, and enrollment coordinators then approached parents for informed consent. Enrolled infants were randomly assigned in a 1:1 ratio to receive standard, clinically determined tests (controls) or standard clinical tests plus trio (infants and parents where available) rWGS for etiologic diagnosis of infants’ underlying conditions (cases; Fig. 1). Randomization was performed automatically by the RAND function in Microsoft Excel at enrollment. Parents and clinicians were initially blinded. However, by day ten they were notified of randomization assignment, to minimize parental anxiety and allow consideration of crossover to rWGS. The study design was adaptive, with modification of enrollment prospectively planned following interim data analysis after approximately 2 years of accrual. ABSTRACT.SAMPLE SIZE DETERMINATION: The study proposed a sample size of 500 in each group (1000 total), with 82% power to detect a difference of 0.05 in the proportion of molecular diagnoses (using a case diagnosis proportion of 0.1 and a control diagnosis proportion of 0.05; two group, continuity-corrected χ2 test with a 0.05 two-sided significance level). We assumed that both primary outcome groups were independent, and contained 500 subjects. We are also interested in comparing the mean time to molecular diagnosis among the two independent study arms. Assuming that 5% of control subjects and 10% of those receiving rWGS receive a molecular diagnosis, the study had more than 98% power to detect the difference in mean time to molecular diagnosis between the cases and controls (96 ± 24 h in rapid whole-genome sequencing cases vs. 240 ± 72 h in controls; two group Satterthwaite t-test with a 0.05 two-sided significance). ABSTRACT.ASCERTAINMENT OF CLINICAL FEATURES AND STUDY MEASURES: The clinical features of affected infants receiving rWGS were ascertained comprehensively by review of the electronic medical record and discussion with physicians and entered in a study REDCap database.56 Phenotypic features were translated into Human Phenotype Ontology terms and mapped to ~5000 monogenic diseases with the clinicopathologic correlation tools SSAGA, Phenomizer and Phenolyzer, generating rank-ordered, deep differential diagnosis lists27,57, 58 (Table S1). Baseline demographics including age, gender, gestational age, birth weight, APGAR scores, and family history were collected. Other study measures were entered into the REDCap database, including diagnostic tests ordered during hospitalization, changes in clinical management following diagnostic test reporting, length of hospitalization, and mortality. Enrollment was from October 2014 to June 2016, and data collection continued until November 2016. ABSTRACT.TRIAL OVERSIGHT: The investigators designed the trial in consultation with NICU and PICU staff and program managers of the funding agencies, the National Human Genome Research Institute (NHGRI) and Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD).24 The investigators received a pre-submission opinion from the Food and Drug Administration (FDA), Center for Devices and Radiological Health (CDRH), Office of In Vitro Diagnostics and Radiological Health (OIR), that the study posed a nonsignificant risk for enrollees, and did not need to be performed under an Investigational Device Exemption (FDA/CDRH/OIR submission Q140271, May 8, 2014). The study was approved by the Institutional Review Boards at Children’s Mercy—Kansas City (CM-KC) and Rady Children’s Hospital, San Diego, and conducted in accordance with the Declaration of Helsinki. Data were collected and analyzed by the investigators. All authors participated in the writing of the manuscript and approved the draft that was submitted for publication. The funding sources were not involved in the collection, analysis, or interpretation of the data, or the writing of the report. The first draft of the manuscript was written by the corresponding author. The authors vouch for the accuracy and completeness of the data and data analyses and for the fidelity of the trial to the protocol (ClinicalTrials.gov accession NCT02225522). ABSTRACT.RAPID WHOLE-GENOME SEQUENCING: rWGS was performed under a research protocol and employed 26-h–7-day methods, guided by acuity of illness of the proband as described.5,6,20, 27 When possible, rWGS was performed on specimens from both biological parents and affected infants simultaneously. Of 37 infants receiving rWGS, 31 were analyzed as trios, 3 as mother-infant duos, 2 as singletons, and 1 as a quad with two affected siblings. Genomic DNA was prepared for rWGS using either TruSeq PCR Free (Illumina, San Diego) or KAPA HYPER (KAPA Biosystems), and resultant libraries were quantified by real-time PCR. WGS was with the Illumina HiSeq 2500 (v4 chemistry, 2 × 125 or 2 × 101 nucleotides, nt) in rapid run or high output mode, or HiSeq 4000 (2 × 125 nt). rWGS was to a minimum depth of 90 Gb per genome, and the average genome coverage was 40-fold (Table S2, Figure S1). All samples met established quality metrics. ABSTRACT.RAPID WGS ANALYSIS AND DIAGNOSTIC INTERPRETATION: rWGS were generated with Illumina RTA 1.12.4.2 and CASAVA-1.8.2, and aligned to the human reference genome GRCh37.p5 using GSNAP and bwa-mem v0.7.12. Nucleotide (nt) variants were detected and genotyped with the Genome Analysis Tool Kit (v1.6-13. and v3.2-2).5,6,20, 27 Copy number variants and structural variants were not detected in WGS. Nucleotide variants were annotated with the Rapid Understanding of Nucleotide variant Effect Software (RUNES).5,6, 20–27 Variants were interpreted by board certified molecular geneticists using American College of Medical Genetics guidelines for pathogenic and likely pathogenic classifications.59 Causative variants were identified primarily with Variant Integration and Knowledge INterpretation in Genomes (VIKING) software.5, 6 Inputs for VIKING were the annotated genomic variant file produced by RUNES and a Phenomizer file of the genes on the comprehensive differential diagnosis.5, 660 Expert interpretation and literature curation were performed for likely causative variants with regard to evidence for pathogenicity. The FDA and IRB approved return of verbal, provisional rWGS diagnosis to the treating physician in exceptional cases, where the results were actionable and the infant was imminently likely to die or have worsening morbidity. Familial relationships were confirmed by segregation analysis of variants. All diagnostic genotypes were confirmed by Sanger sequencing prior to final reporting. rWGS and Sanger sequencing were performed in a laboratory licensed by the Clinical Laboratory Improvement Amendments and accredited by the College of American Pathologists. Additional expert consultation and functional confirmation were performed in selected cases when the subject’s phenotype differed from previous mutation reports for that disease gene or when the pathogenicity of variants was uncertain. In the absence of a diagnosis, a research note was placed in the medical record to indicate that testing was complete. At time of study performance, clinical grade detection of copy number and structural variants was not performed. Secondary and incidental findings were not reported. ABSTRACT.STANDARD GENETIC TESTING: Standard clinical testing for genetic disease etiologies was performed in infants based on physician clinical judgment, assisted by subspecialist recommendations. Specimens for all standard tests were collected and transported as quickly as possible, and all standard tests were performed as expeditiously as possible. The set of genetic tests considered to be standard was developed by three pediatrician subspecialists. Standard genetic tests were those ordered through the electronic medical record, and included biochemical and immunologic testing for genetic diseases, array comparative genomic hybridization, fluorescence in situ hybridization, high resolution chromosomes analysis, Sanger sequencing of genes, non-expedited proband targeted next-generation sequencing (NGS) gene panels, non-expedited proband whole-exome sequencing (WES), non-expedited proband WGS, methylation studies, and gene deletion/duplication assays (Table S3), as well as Kansas or Missouri state newborn screening (including five lysosomal storage diseases). ABSTRACT.TRIAL END POINTS: The pre-specified primary end point was a comparison of the proportion of patients receiving a molecular diagnosis within 28 days of enrollment. The pre-specified secondary end points were the proportion of patients receiving a molecular diagnosis within the neonatal period (28 days of life), total diagnostic rate, time-to-diagnosis, and percentage of patients with a change in management related to test results in the two arms. Other pre-specified end-points were the length of hospitalization and short term mortality rate. Data related to these end-points were entered into the REDCap database and audited by two study investigators. The end-points were not changed after the trial commenced. Change in management was determined by a survey of nominating clinicians and review of the electronic health record by at least two pediatrician subspecialists with substantial expertise in genomic medicine to identify change in treatments and procedures, canceled tests, new focused tests, recommendation for specific follow-up related to the diagnosis, and changes in consultation related to the diagnosis.61 A modified Delphi method was used to determine inclusion of change in management where there was disagreement. ABSTRACT.STATISTICAL ANALYSIS: Statistical analyses were based on the intention-to-treat principle to avoid confounding due to the crossover of controls to the rWGS group. Variables representing whether patients received a diagnosis and whether they received a change in care were treated as dichotomous (y/n). Controls were considered diagnosed only if they received a molecular diagnosis from standard tests regardless of whether they were crossed over and received a diagnosis from rapid WGS. Cases were classified as diagnosed if they received at least one diagnosis from either rapid WGS or standard tests. Primary analyses comparing 28-day diagnostic rates between study arms were conducted using Fisher’s exact test. Differences in secondary endpoints, including total diagnoses, clinical utility of diagnoses, and diagnoses before discharge, were also assessed with Fisher’s exact test. A two-sample t-test was performed to evaluate the null hypothesis of no difference in mean age at hospital discharge for rapid WGS cases and controls. Kaplan–Meier analyses were used to compare time to diagnosis for each study arm. We generated plots of the cumulative diagnostic rates, estimated as one minus the Kaplan–Meier function, and 95% confidence intervals. For the analysis of age at diagnosis, time was measured from date of birth to date of diagnosis. For the analysis of time to diagnosis from first test, time to diagnosis was measured from the date of enrollment. Patients that did not receive a diagnosis by the end of the study had their data censored at the final date of data collection (November 2016). In general, the log-rank test is most powerful in the presence of proportional hazards. Thus for age at death, for which there was no evidence of non-proportional hazards, the log-rank test was performed.62 When there was evidence of a non-constant hazard ratio, between-group differences were evaluated with the Peto-Peto test.63, 64 The latter was used as an alternative to the log-rank test when comparing time to diagnosis between WGS plus standard tests and standard tests alone because of its increased power, especially when differences are hypothesized to occur early in time. As a sensitivity analysis, we reclassified patients who received a partial diagnosis as undiagnosed and repeated analyses for relevant primary and secondary end-points. All reported p-values are two-sided and were considered statistically significant if less than 0.05. Statistical analyses were performed in R version 3.3.0.65 ABSTRACT.DATA AND MATERIAL AVAILABILITY: Data are available at LPDR (https://www.nbstrn.org/research-tools/longitudinal-pediatric-data-resource). ABSTRACT.KEY POINTS: Question: What proportion of acutely ill inpatient infants receive a diagnosis of a genetic disease within 28 days with rapid whole-genome sequencing? Findings: In a RCT of 65 infants, the diagnostic sensitivity of rapid whole-genome sequencing within 28 days was 31 vs. 3% with standard genetic testing, a significant difference. Meaning: In NICU and PICU infants with diseases of unknown etiology, rapid whole-genome sequencing may be warranted as a first-line diagnostic test. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary Material 1

TITLE: Does offering an incentive payment improve recruitment to clinical trials and increase the proportion of socially deprived and elderly participants? ABSTRACT.BACKGROUND: Patient recruitment into clinical trials is a major challenge, and the elderly, socially deprived and those with multiple comorbidities are often underrepresented. The idea of paying patients an incentive to participate in research is controversial, and evidence is needed to evaluate this as a recruitment strategy. ABSTRACT.METHOD: In this study, we sought to assess the impact on clinical trial recruitment of a £100 incentive payment and whether the offer of this payment attracted more elderly and socially deprived patients. A total of 1,015 potential patients for five clinical trials (SCOT, FAST and PATHWAY 1, 2 and 3) were randomised to receive either a standard trial invitation letter or a trial invitation letter containing an incentive offer of £100. To receive payment, patients had to attend a screening visit and consent to be screened (that is, sign a consent form). To maintain equality, eventually all patients who signed a consent form were paid £100. ABSTRACT.RESULTS: The £100 incentive offer increased positive response to the first invitation letter from 24.7% to 31.6%, an increase of 6.9% (P < 0.05). The incentive offer increased the number of patients signing a consent form by 5.1% (P < 0.05). The mean age of patients who responded positively to the invitation letter was 66.5 ± 8.7 years, whereas those who responded negatively were significantly older, with a mean age of 68.9 ± 9.0 years. The incentive offer did not influence the age of patients responding. The incentive offer did not improve response in the most socially deprived areas, and the response from patients in these areas was significantly lower overall. ABSTRACT.CONCLUSION: A £100 incentive payment offer led to small but significant improvements in both patient response to a clinical trial invitation letter and in the number of patients who consented to be screened. The incentive payment did not attract elderly or more socially deprived patients. ABSTRACT.TRIAL REGISTRATIONS: Standard care versus Celecoxib Outcome Trial (SCOT) (ClinicalTrials.gov identifier: NCT00447759). Febuxostat versus Allopurinol Streamlined Trial (FAST) (EudraCT number: 2011-001883-23). Prevention and Treatment of Hypertension with Algorithm Guided Therapy (British Heart Foundation funded trials) (PATHWAY) 1: Monotherapy versus dual therapy for initiating treatment (EudraCT number: 2008-007749-29). PATHWAY 2: Optimal treatment of drug-resistant hypertension (EudraCT number: 2008-007149-30). PATHWAY 3: Comparison of single and combination diuretics in low-renin hypertension (EudraCT number: 2009-010068-41). ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13063-015-0582-8) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Efficient recruitment of patients into clinical trials is a major challenge in medical research. Recruited patients are often those interested in their health. The elderly, socially deprived and those with significant comorbidities are generally underrepresented. This raises concerns that the results of clinical trials may not be generalisable to all groups in the wider population and questions about the validity of using outcomes from these trials when making clinical decisions affecting the broader population [1,2]. Various methods have been used to improve recruitment into clinical trials. Recruitment strategies depend largely on the type of trial and the patient population required. Recruitment into studies with stringent inclusion and exclusion criteria can be particularly challenging. Traditional recruitment methods, including use of primary and secondary care practitioners, remain important; however, Investigators have also used public awareness campaigns and advertising through various media outlets to promote the benefits to society of participating in research and to attract a wider variety of patients [3-5]. The concept of using financial incentives to recruit patients into clinical trials is controversial, and attitudes toward them vary between countries and cultures. In the United Kingdom, healthy volunteers participating in research may be paid for their services [6]. For patients participating in clinical trials, there is agreement that they should be reimbursed for reasonable expenses; however, there is a clear distinction between appropriate reimbursement and additional payment as a financial incentive to participate. The situation is different in the United States, which has a largely insurance-based health care system. There, financial incentives are frequently used. A 2002 report found that in 32 US health care organisations surveyed, 58% of patients were paid an incentive to participate in research [7]. Researchers in the United States found that moderate incentive payments were effective at improving recruitment and were not seen as undue or unjust inducements [8]. Before further debate on the ethics of offering incentive payments to patients to participate in research, we felt it important to assess the effectiveness of incentive payments as a recruitment strategy in Scotland, both in terms of absolute numbers recruited and in terms of widening the demographic profile of those screened. BODY.METHODS: We received ethical approval from the Scotland Research Ethics Committee (REC 12/SS/0006) to assess whether the offer of a fixed payment of £100 would improve recruitment into five different clinical trials currently running in the United Kingdom. The trials studied were the Standard care versus Celecoxib Outcome Trial (SCOT) [9], the Febuxostat versus Allopurinol Streamlined Trial (FAST) and the three British Heart Foundation–funded Prevention and Treatment of Hypertension with Algorithm Guided Therapy (PATHWAY) studies (1, 2 and 3). All trial participants provided us their written, informed consent to participate. Further details of the trials are shown in Table 1.Table 1 Overview of the five clinical trials used in this study a Trial Sponsor Outline SCOT [10] (ClinicalTrials.gov identifier: NCT00447759) University of Dundee Trial comparing the cardiovascular safety of celecoxib with that of other traditional NSAIDs in patients older than 60 years of age who are taking long-term NSAIDs for arthritis FAST (EudraCT number: 2011-001883-23) University of Dundee Trial comparing the cardiovascular safety of febuxostat versus allopurinol in patients over the age of 60 years with symptomatic hyperuricaemia PATHWAY 1 (EudraCT number: 2008-007749-29) University of Cambridge Trial of newly diagnosed hypertension in patients aged 18 to 79 years comparing monotherapy with dual therapy as initial hypertension treatment PATHWAY 2 (EudraCT number: 2008-007149-30) University of Cambridge Trial investigating treatment of resistant hypertension in patients aged 18 to 79 years with uncontrolled blood pressure on three anti-hypertensive agents PATHWAY 3 (EudraCT number: 2009-010068-41) University of Cambridge Trial comparing single-agent and combination diuretic therapy for low-renin hypertension in patients aged 18 to 80 years with at least one component of the metabolic syndrome aFAST, Febuxostat versus Allopurinol Streamlined Trial; NSAID, Non-steroidal anti-inflammatory drug; PATHWAY, Prevention and Treatment of Hypertension with Algorithm Guided Therapy, British Heart Foundation–funded trials; SCOT, Standard care versus Celecoxib Outcome Trial. For each of the five trials, potentially suitable patients were identified through a search of their general practitioners’ (GPs’) practice databases and invited to a screening visit. The invitation letters contained either the offer of the £100 incentive payment if the patient consented to be screened or a standard invitation letter with no incentive offer. If there was no response to the first invitation letter, a further letter was sent offering the incentive. Sample size calculations were based on previous recruitment rates calculated from available data and differed between the five trials. For the FAST and SCOT studies combined, to detect a 50% increase in response to invitation letters at 80% power required 225 letters to be sent for each group. For the three PATHWAY studies, to detect a 50% increase would require 424 letters in each group; therefore, for practicality, it was decided to calculate for a 100% increase in response at 80% power, which required 121 invitation letters to be sent for each group. Owing to the difficulties in determining what would be considered a worthwhile increase in recruitment balanced against the cost of the incentive payment, sample size calculations were pragmatic. To receive the payment, the patient had to attend a screening visit and consent to be screened for the trial (that is, sign a consent form). To maintain equity, the £100 incentive was paid to all patients who signed a consent form for any of the studies (without regard to whether the incentive offer was in the invitation letter). Eligible patients who wished to receive the payment provided bank details to the finance department of the Medicines Monitoring Unit of the University of Dundee, and the £100 payment was transferred directly into the patient’s bank account after the signed consent form was received. Patients were randomised centrally from the Medicines Monitoring Unit of the University of Dundee. A list of suitable patients from each GP practice was generated by the research nurses for each trial. Patients on this list were randomly assigned a 0 (no incentive offer) or a 1 (incentive offer) using a computer algorithm (rand.nextdouble() > 0.500). This was a simple fixed randomisation method rather than an adaptive method; therefore, no effort was made to balance the groups. The code used generated random figures from ≥0 to <1. Figures ≤0.5 were assigned a 0, and figures >0.5 were assigned a 1. This led to a small unforeseen bias towards generating a 0 (no incentive offer). This imbalance was not evident until after the trial had been completed. Research nurses and study personnel were not blinded to the randomisation. Allocation of the incentive offer was independent for each trial. Basic demographic information was recorded for each patient who was sent an invitation letter, including age, sex and postcode. The Scottish Index of Multiple Deprivation (SIMD) 2012 based on postcode was used as a measure of socioeconomic status [11]. The SIMD combines 38 indicators across 7 domains, including income, employment, health, education, skills, housing, access and crime. Although SIMD is not a perfect indicator, it was considered a reasonable way of estimating socioeconomic status for our trial populations, given the data we had available. The primary outcome for this study was patient response to the first invitation letter, depending on whether the patient was offered the £100 incentive. Secondary outcomes included a comparison of the demographics of patients who responded positively between the incentive and non-incentive groups and the number of patients in each group who consented to be screened (and therefore received the incentive payment) and eventually were randomised into each study. The response to the follow-up letter was analysed separately. A study schematic with patient numbers is shown in Figure 1.Figure 1 Study schematic. GP, General practitioner practice; FAST, Febuxostat versus Allopurinol Streamlined Trial; PATHWAY, Prevention and Treatment of Hypertension with Algorithm Guided Therapy, British Heart Foundation–funded trials; SCOT, Standard care versus Celecoxib Outcome Trial. Data were summarised as mean and standard deviation for continuous variables and number of patients (percent) for categorical variables. The χ2 test and an independent t-test were performed to determine significant differences. Response rates to the first invitation letter were compared between the patients who were offered the incentive and patients who were not offered the incentive. A logistic regression model was employed to access factors affecting positive responses to the first invitation letter. Analysis was undertaken using SAS version 9.2 software (SAS Institute, Cary, NC, USA). BODY.RESULTS: A total of 1,015 patients were sent a first invitation letter for one of the five clinical trials. This total comprised 332 (32.7%) patients for FAST, 181 (17.8%) patients for SCOT, 93 (9.2%) patients for PATHWAY 1, 210 (20.7%) patients for PATHWAY 2 and 199 (19.6%) patients for PATHWAY 3. A total of 481 (47.4%) patients were offered the incentive in the first invitation letter. The mean age of invited study subjects was 66.3 ± 9.9 years, and 58% of the patients were male. All patients were recruited in the East of Scotland (Dundee, Fife and Perth). There were no differences in age, sex, social deprivation, geographic location and invitation to different trials between the incentive and non-incentive groups (Table 2).Table 2 Patient characteristics a Offered incentive ( N  = 481) Not offered incentive ( N  = 534) Age (mean, SD) 66.2 (10.2) 66.3 (9.6) Sex  Male 281 (58.4) 308 (57.7)  Female 162 (33.7) 182 (34.1)  Unknown 38 (7.9) 44 (8.2) SIMD deprivation categoryb  1–3 (least deprived) 138 (28.7) 134 (25.1)  4–7 242 (50.3) 274 (51.4)  8–10 (most deprived) 101 (21.0) 125 (23.5) Geographic areasb  Angus and Dundee 217 (45.1) 215 (40.3)  Fife 200 (41.6) 234 (43.9)  Perth 64 (13.3) 84 (15.8) Target patient group  FAST 158 (32.9) 174 (32.6)  SCOT 84 (17.5) 97 (18.2)  PATHWAY 1 46 (9.6) 47 (8.8)  PATHWAY 2 101 (21.0) 109 (20.4)  PATHWAY 3 92 (19.1) 107 (20.0) aFAST, Febuxostat versus Allopurinol Streamlined Trial; PATHWAY, Prevention and Treatment of Hypertension with Algorithm Guided Therapy, British Heart Foundation–funded trials; SCOT, Standard care versus Celecoxib Outcome Trial; SIMD, SD, Standard deviation; Scottish Index of Multiple Deprivation. Data are mean (SD) or number (%). There were no significant differences between groups bIncludes one patient with missing data. BODY.PRIMARY OUTCOME: RESPONSE TO FIRST INVITATION LETTER: The response rates to the first invitation letter were 284 (28.0%) positive responses, 279 (27.5%) negative responses and 452 (44.5%) patients who did not respond at all. Table 3 shows the differences in positive responses between the incentive and non-incentive groups for each trial. Overall figures show there was a 6.9% increase in positive responses with the incentive offer (95% confidence interval (CI), 1.35 to 12.40; P = 0.04); however, there were marked differences in response rates for each of the five trials.Table 3 Invitation to screening visit and outcome of first invitation letter, by trial a FAST ( N  = 332) SCOT ( N  = 181) PATHWAY 1 ( N  = 93) PATHWAY 2 ( N  = 210) PATHWAY 3 ( N  = 199) Overall ( N  = 1,015) Offered incentive payment, n (%) 158 (47.6%) 84 (46.4%) 46 (49.5%) 101 (48.1%) 92 (46.2%) 481 (47.4%) Not offered incentive payment, n (%) 174 (52.4%) 97 (53.6%) 47 (50.5%) 109 (51.9%) 107 (53.8%) 534 (52.6%) Number of responses to first invitation letter Positive  Incentive offer 68 (43.0%) 34 (40.5%) 5 (10.9%) 19 (18.8%) 26 (28.3%) 152 (31.6%)  No incentive offer 54 (31.0%) 31 (32.0%) 7 (14.9%) 19 (17.4%) 21 (19.6%) 132 (24.7%)   % change with Incentive ((95% CI)) 12.0% 8.5% −4.0% 1.4% 8.7% 6.9%* (1.35 to 12.40) Negative  Incentive offer 40 (25.3%) 15 (17.9%) 14 (30.4%) 30 (29.7%) 23 (25.0%) 122 (25.4%)  No incentive offer 49 (28.2%) 16 (16.5%) 16 (34.0%) 37 (33.9%) 39 (36.4%) 157 (29.4%)   % change with incentive ((95% CI)) −2.9% −1.4% −3.6% −4.2 −11.4% −4.0% (−1.47 to 9.47) No response  Incentive offer 50 (31.6%) 35 (41.7%) 27 (58.7%) 52 (51.5%) 43 (46.7%) 207 (43.0%)  No incentive offer 71 (40.8%) 50 (51.5%) 24 (51.1%) 53 (48.6%) 47 (43.9%) 245 (45.8%)   % change with incentive ((95% CI)) −9.2% −9.8% 7.6% 2.9% 2.8% −2.8% (−3.27 to 8.92) Number of patients signing a consent form Incentive offer 58 (36.7%) 26 (30.9%) 3 (6.5%) 4 (4.0%) 9 (9.8%) 100 (20.8%) No incentive offer 41 (23.6%) 24 (24.7%) 4 (8.5%) 9 (8.3%) 6 (5.6%) 84 (15.7%) % change with incentive ((95% CI)) 13.1% 6.2% −2.0% −4.3% 4.2% 5.1%* (0.31 to 9.85) Number of patients randomised into trial Incentive offer 58 (36.7%) 26 (30.9%) 2 (4.3%) 3 (3.0%) 5 (5.4%) 94 (19.5%) No incentive offer 40 (23.0%) 24 (24.7%) 3 (6.4%) 6 (5.5%) 0 73 (13.7%) % change with incentive ((95% CI)) 13.7% 6.2% −2.1% −2.5% 5.4%* 5.9%* (1.30 to 10.49) aCI, Confidence interval; FAST, Febuxostat versus Allopurinol Streamlined Trial; PATHWAY, Prevention and Treatment of Hypertension with Algorithm Guided Therapy, British Heart Foundation–funded trials; SCOT, Standard care versus Celecoxib Outcome Trial. *P < 0.05. A logistic regression model based on age, sex, incentive payment, trial, deprivation decile and geographic area showed that the incentive payment, age and invitation to the PATHWAY trials were significantly associated with response rates. Older patients and those invited to the PATHWAY trials were more likely to answer negatively to the first invitation letter (adjusted odds ratio, 0.95 (95% CI, 0.93 to 0.97) for age; 0.16 (0.07 to 0.39) for PATHWAY 1; 0.26 (0.14 to 0.50) for PATHWAY 2 and 0.29 (0.15 to 0.56) for PATHWAY 3). BODY.SECONDARY OUTCOMES: BODY.PATIENT DEMOGRAPHICS: The mean ages in years for the positive, negative and no-response groups for all trials are shown in Figure 2. Older patients were significantly more likely to respond negatively to the invitation letter, regardless of whether they were offered the incentive (P < 0.05). There are limitations in combining all trials for age, as the FAST and SCOT trials recruited only patients over the age of 60, whereas the PATHWAY trials were open to patients as young as 18 years of age. The oldest patients were in the FAST trial, with a mean age of 71.5 ± 7.6 years, and the youngest patients were in the PATHWAY 1 study, with a mean age of 57.9 ± 11.9 years. However, within each trial, older patients were numerically more likely to respond negatively to the first invitation letter; this reached statistical significance for the FAST, PATHWAY 1 and PATHWAY 3 studies. None of the trials showed that the offer of the incentive affected the age of the patients responding. Full results for the effect of age on responses by trial are shown in Additional file 1: Table S1.Figure 2 Bar graph showing mean age (in years) of patients who responded positively versus negatively to the first invitation letter with versus without the incentive offer. Patients from more deprived areas (SIMD deciles 1–3) were less likely to respond positively to the invitation letter, with an overall positive response rate of 21.3% compared to an overall positive response of 31.4% from patients in the least deprived areas (SIMD deciles 8–10) (P = 0.032). There were no significant differences in positive response to the first invitation letter with or without the incentive offer in the most deprived areas or in the least deprived areas; however, patients in SIMD deciles 4–7 were significantly more likely to respond positively to the incentive offer (37.2% versus 24.0%) (P = 0.004). Full results are available in Additional file 2: Table S3. BODY.PATIENTS CONSENTED AND RANDOMISED: In total, 284 patients responded positively to the first invitation letter, and 184 (64.8%) of these signed a consent form (making them eligible for the incentive payment). Of these 184 patients, 100 who signed a consent form were offered the incentive, which represents a 5.1% increase in consented patients who received the incentive offer (P = 0.037). Of the 184 consented patients, 167 were ultimately randomised into a trial. Table 3 shows the differences in consented and randomised patients by trial. The greatest (although not statistically significant) positive changes with the incentive offer were seen in the FAST trial. BODY.RESPONSE TO NON-RESPONDER LETTERS: Non-responder letters were sent to 374 of the 452 non-responders to the first letter (full results are available in Additional file 3 BODY.OVERALL OUTCOMES FOR ALL PATIENTS: Final figures for both the first and the non-responder letters show that, in total, there were 316 patients who responded positively to the invitation letter (184 offered the incentive, 132 not offered the incentive). Of these 316 patients, 251 attended a screening visit. A total of 202 patients signed a consent form and were eligible for the £100. Ultimately, 181 patients were randomised into a study (104 for FAST, 58 for SCOT, 5 for PATHWAY 1, 9 for PATHWAY 2 and 5 for PATHWAY 3). Thus, after writing to 1,015 patients, 181 were ultimately randomised into a trial, giving an overall randomisation rate of 17.8%. BODY.COST-EFFECTIVENESS: The additional cost incurred in undertaking this incentive payment trial was £100 per patient who signed a consent form, as invitation letters, trial information and screening visits were unchanged from the usual recruitment process of each trial. With 202 patients consented into the trials, the total cost of the payments to patients in the trial was £19,900 (3 patients did not accept the incentive payment). The cost to each trial of paying a £100 incentive is determined by the number of patients signing the consent form (as only these patients are paid). Some patients who responded positively to the invitation letter did not meet trial inclusion criteria, however; therefore, despite their wish to participate and claim the £100, they were ineligible. Table 4 shows the cost for each additional patient who responded positively to the invitation letter, as well as the cost for each consenting patient if the incentive was offered. Overall, the cost was £1.549 to get one additional patient to respond positively; however, significant differences between the trials were again evident.Table 4 Cost per additional responding or consented patient from first invitation letter by trial a Increase in positive response with incentive Cost per additional patient Increase in consented patients with incentive Cost per additional consented patient Overall 6.9% £1,549 5.1% £1,961 FAST 12.0% £933 13.1% £763 SCOT 8.5% £1,276 6.2% £1,613 PATHWAY 1 −4.0% N/A −2.0% N/A PATHWAY 2 1.4% £7,243 −4.3% N/A PATHWAY 3 8.7% £1,249 4.2% £2,381 aFAST, Febuxostat versus Allopurinol Streamlined Trial; N/A, Not applicable; PATHWAY, Prevention and Treatment of Hypertension with Algorithm Guided Therapy, British Heart Foundation–funded trials; SCOT, Standard care versus Celecoxib Outcome Trial. BODY.DISCUSSION: Over one-third of the patients invited to participate in a clinical trial did not respond at all to the invitation letter. This lack of response is commonplace when attempting to recruit participants for research and means effective clinical trial recruitment is difficult. The offer of a £100 incentive payment did have some impact in improving patient response to the first invitation letter, and positive responses increased by 6.9% in the incentive group. The improvement in initial positive response did lead to a small (5.1%) increase in the number of patients signing a consent form. These increases were statistically significant when looking at only the response to the first invitation letter, but they became non-significant when overall figures for both the first and non-responder letters were used, owing to the poor response to the non-responder letters. The five trials included in this study targeted different patient populations and required different levels of input from recruited patients; therefore, response rates varied significantly between the different trials. The recruitment for the SCOT and FAST trials was through GP’s writing to patients, and participants had to be older than 60 years of age and meet other entry criteria regarding their medication and medical history. SCOT and FAST were streamlined trials with the aim of replicating day-to-day clinical care; therefore, patients attended a screening visit and were then followed up remotely, so the burden on participants was relatively small. The PATHWAY studies recruited patients from primary care who were aged 18–80 years with either newly diagnosed or partially treated hypertension. All three PATHWAY studies involved multiple patient visits to the study centre as well as home blood pressure monitoring. The PATHWAY trials therefore often targeted a younger, working patient group and required a significantly greater investment in terms of time and effort from participants. Overall, initial positive response rates to SCOT and FAST invitations (both with and without the incentive) were 37% and 36%, respectively, compared to 19% for the PATHWAY trials combined. The greatest increase in patients actually randomised into a trial with the incentive offer was seen for the FAST trial, but this 13.7% increase was not statistically significant. Subsequent screening and randomisation rates were also significantly poorer for all of the PATHWAY studies, which was due to stringent study entry criteria for blood pressure as well as the greater perceived burden of multiple study visits for the participants. Recruitment into PATHWAY 1 and 2 appears to have been negatively affected by the incentive offer; however, the number of consented patients was too small to allow meaningful interpretation. Recruitment into PATHWAY 3 increased with the incentive offer, but this still represented only 5 patients randomised out of 199 patients contacted. Cost-effectiveness was calculated as the cost of one additional responding or consented patient if all patients received £100. The FAST trial showed the greatest increase in consented patients with the incentive offer; however, even with a 13.1% increase, the cost per additional patient was still high at £763. It would be beyond the scope of most studies, particularly those lacking commercial funding, to view this as cost-effective. The cost-effectiveness analysis is also perhaps unfair because the calculations were based only on the number of patients who received the payment. Patients who did not meet study inclusion criteria were never in a position to be able to receive the incentive payment, even though they had indicated a willingness to take part in a clinical trial. Therefore, cost-effectiveness figures could be marginally improved if investigators invited fewer unsuitable patients to screening visits. The results of our present study indicate that the offer of a £100 financial incentive had only a limited impact on improving recruitment into the five clinical trials involved. This may initially appear surprising, as most people would acknowledge that we are all driven to some extent by the attainment of financial reward. However, in the health care setting, people are motivated to different degrees by personal gain and altruism. In 1970, Richard Titmuss claimed that paying people for blood donations actually made them less likely to donate, as the payment removed the altruistic incentive [12]. It was then suggested that increasing the payment would be effective; however, subsequent work has shown that payment can reduce intrinsic motivation and that people are often distrustful of payment offered for altruistic behaviour, including participation in clinical trials [13]. There is significant individual variation in how people respond to financial incentive and complex reasons underpinning these decisions. What may seem a worthwhile reward for some people may not attract others and may even actively discourage people who are uncomfortable with receiving financial reward for altruistic behaviour. The incentive offer for this study was £100. This figure was chosen as a compromise between reasonable compensation for the degree of inconvenience caused against an effective incentive to participate. The level of the incentive offered must fulfil the primary objective of increasing recruitment without being seen as financial coercion. Studies done in the United States have shown that the level of payment does influence response. For example, in a study in which researchers looked at enrolling teenagers in a smoking cessation program, the results showed that any incentive was better than no incentive and that a $15 cash incentive improved responses compared to a $2 cash incentive and entry into a $200 prize drawing [3,10]. Incentive payments in clinical research are controversial, particularly regarding where to draw the line between financial incentive and financial coercion. This concern is especially relevant when trying to recruit socially deprived and vulnerable members of society [14]. There has also been debate over whether the size of the payment should reflect the risk or inconvenience of the trial and if a standard formula could be developed to determine the amount of the incentive [15,16]. The five trials in this study were all considered low-risk (all used medicines within their licensed indications), and therefore no assessment was made of the participants’ views of the risks involved and whether this influenced their decision to participate. Opinions are divided on this topic, with many people feeling that higher-risk trials should not offer incentive payments at all [17]. Researchers in the United States have looked at how patients respond to payment offers in terms of both the level of payment and the perceived risk associated with the trial. Halpern and colleagues conducted a study where 126 hypertensive patients were provided with information on a series of potential trials of a new anti-hypertensive medication. In a 3 × 3 design, the risk of adverse events and the payment offer ($100, $1,000 or $2,000) were varied. In 34% of patients, the level of payment significantly influenced their willingness to participate. Unsurprisingly, trials with a higher risk of side effects and lower payment offers decreased willingness to participate. They also found a non-significant trend towards wealthier people being more strongly influenced by payment, but they did not find that patients’ perceptions of the risks associated with the trials were altered by the level of the payment offered [8]. Our study shows that, though there may be some case to be made for incentive payments’ marginally improving recruitment into some trials, there was no evidence that the incentive payment broadened the demographics of those participating. The incentive payment offer had no impact on the age of those responding, and, for every trial within the study, there was a trend towards older patients responding negatively to the invitation letter and younger patients not responding at all (irrespective of the incentive offer). This may reflect younger patients having work and family commitments that prevent their participation and older patients having different priorities, such as increased frailty that prevents multiple study visits and presence of multiple comorbidities that make these patients less willing to spend additional time with health care professionals. In terms of social deprivation, the initial positive response (with or without the incentive offer) from patients in the most deprived areas (SIMD deciles 1–3) was 21.3%, which was lower than the overall trial average of 28.0%. Positive responses from patients in the least deprived areas (SIMD deciles 8–10) were higher than the trial average at 30.7%. Patients in the middle deprivation deciles (SMID deciles 4–7) were the only group to show a significant increase in positive responses associated with the incentive payment. We can only speculate on the reasons for this. It might be that the most deprived in society are the least engaged with health care, whereas those who are better off would not be enticed by a relatively small payment but take a greater interest in their health. There are limitations to this trial, including looking at patient recruitment from part of eastern Scotland only. Using five diverse clinical trials complicated interpretation of the results, as recruitment varied widely between trials. There are likely to have been different factors affecting recruitment within each trial, including how the trial was advertised and how patients were approached. It might have been interesting to look at different levels of payment; however, it would be expected that a lower figure would have made even less impact on patient recruitment and that a higher figure would mean the trial was not financially viable. Future work in this area could look at incentives that are not purely financial and are individualised to patients or trials. BODY.CONCLUSIONS: Both simple and complex messages emerge from this study. Put simply, paying patients £100 did entice more people to respond positively to an invitation letter and did result in slightly more randomised patients, particularly in the FAST trial; however, this effect was marginal. Response rates varied a great deal between the different trials, and, even where a significant improvement was observed, it would be a stretch to see this as a cost-effective recruitment method. The incentive payment did not attract the elderly or the more socially deprived. What motivates people to participate in clinical research remains elusive to the research community, and it would appear that £100 is not sufficient motivation for most.

TITLE: Upper secondary school students’ compliance with two Internet-based self-help programmes: a randomised controlled trial Psychiatric symptoms and stress are on the increase among Swedish adolescents. We aimed to study the potential effect and feasibility of two Internet-based self-help programmes, one mindfulness based (iMBI) and the other music based in a randomised controlled trial that targeted adolescents. A total of 283 upper secondary school students in two Swedish schools were randomised to either a waiting list or one of the two programmes, on their own incentive, on schooltime. General psychiatric health (Symptoms Checklist 90), sleep quality (Pittsburgh Sleep Quality Index), and perceived stress (Perceived Stress Scale) were assessed before and after the interventions. In total, 202 participants answered the questionnaires. Less than 20 logged into each intervention and only 1 performed a full intervention (iMBI). No significant differences in any of the scales were found between those who logged in and those who did not. The potential effect of Internet-based self-help programmes was not possible to examine due to low compliance rates. Adolescents seem to have a very low compliance with Internet-based self-help programmes if left to their own incentive. There were no associations between the psychiatric and stress-related symptoms at baseline and compliance in any of the intervention groups, and no evidence for differences in compliance in relation to the type of programme. Additional studies are needed to examine how compliance rates can be increased in Internet-based self-help mindfulness programmes in adolescents, as the potentially positive effects of mindfulness are partly related to compliance rates. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00787-017-1035-6) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Psychiatric symptoms and perceived stress have increased among Swedish adolescents in recent decades [1–3]. Furthermore, there is evidence of significant associations between stress and psychopathology in children and adolescents from both cross-sectional [4, 5] and longitudinal studies [6–9]. Psychiatric symptoms and stress in adolescents can result in negative effects on health, well-being, and academic achievement [10], and seem to be independent of both the academic proficiency and urbanicity of the school [11]. In addition, there is a well-established association between reduced sleep quality and psychiatric symptoms in adolescents. Low sleep quality in adolescents is associated with behavioural problems and impairments in cognitive functioning and mood [12–14], which partly could explain the suggested increase in psychiatric symptoms and stress in Swedish adolescents over time [1, 15]. Internet-based psychotherapy (iPT) seems to be a promising treatment alternative in targeting psychiatric health problems; iPTs are relatively easy to access and could provide therapy to many people at a low cost [16]. This form of therapy may be particularly suitable for adolescents, since young people in general are familiar with using the Internet and it could also help many adolescents who refrain from seeking help in regular health care [17]. The effects of, as well as the compliance to, iPTs in adolescents remain, however, largely unstudied as very few studies have been based on younger participants [18]. Mindfulness-based interventions (MBIs) have been adapted to the Internet as a vehicle of delivery and could therefore be suitable for adolescents. MBIs are well-renowned and well-validated psychotherapies [19–28], and a meta-analysis in non-clinical settings concluded that mindfulness-based stress reduction (MBSR) can reduce stress levels in healthy people [29]. A recent randomised controlled trial (RCT) in Sweden was conducted on adults with anxiety, depression, and stress and adjustment disorders treated in primary health care. The RCT compared group-based mindfulness therapy with individual-based cognitive behavioural therapy (CBT) and found that both therapies resulted in significant decreases in psychiatric symptoms [30]. Mindfulness-based therapies can also be combined with CBT, i.e. mindfulness-based cognitive therapy (MBCT) [31]. Two recent review articles concerning mindfulness on adolescents concluded that it holds promise for students, in terms of improving cognitive performance and resilience to stress [32, 33]. Another study concluded that there are few possibilities to deliver MBIs in schools as few teachers, or other school personnel, are able to provide MBIs to the students [34]. The use of Internet-based MBIs (iMBI) may therefore be promising in terms of accessibility [16]. Several studies on adults have shown promise in using the Internet as a vehicle of psychotherapy delivery [35–40]. A narrative review concluded that iMBIs are feasible in terms of effectiveness, acceptability, and economy, and that further research examining different modes of iMBIs is warranted [41]. To our knowledge, no iMBI study on the potential effects as well as compliance has been carried out on adolescents. However, an Australian qualitative study has proposed iMBI as something desired by many adolescents [42]. Some previous studies have examined the potential effects of iMBI as well as the compliance in adults. The included clinical diagnoses and outcomes were relapse of major depression [39] and depression, anxiety and stress and adjustment disorders [35, 36, 38, 40, 43]. In general, these studies have found positive effects of iMBI, although the compliance has varied. The first aim of this study was to investigate whether there is an effect of iMBI, delivered as an Internet-based self-help programme, on psychiatric and stress-related symptoms in adolescents. The second aim was to investigate the feasibility, in terms of compliance, of the intervention. The third aim was to investigate whether there is any association between psychiatric and stress-related symptoms at baseline and compliance to the intervention in the adolescents. BODY.METHODS: BODY.STUDY POPULATION: In the process of choosing the population, we opted for two different schools in terms of academic performance and urbanicity, to obtain an adequate sample that could be considered relatively representative of Swedish upper secondary school students. This selection is more thoroughly described in the baseline cross-sectional study [11], which represents a larger randomised, controlled, single-blinded study (CHAMPS) aimed to investigate whether iMBI could be used to prevent psychiatric and stress-related symptoms. All Swedish-speaking students enrolled at the schools (adolescents aged 15–19 years) received information, initially through personal letters sent to their home addresses, and thereafter lectures in the schools (10-min oral information from the researchers in groups of 10–100 students with the possibility to ask questions). We had three inclusion criteria: to be able to read and understand Swedish, to have an e-mail address, and to be willing to participate in an 8-week Internet-based self-help programme. A total of 1404 students were attending the two schools during the study period. The Swedish-speaking inclusion criterion excluded one subject, leaving 1403 possible participants. The second inclusion criterion did not lead to exclusion of any potential participants and the third criterion, willingness to participate in a stress reduction intervention, left 283 individuals all of whom gave written informed consent. The 283 participants were numbered consecutively after the arrival of their written consent form and were divided into seven groups, based on the three grades (first to third year of upper secondary school) and sex, to acquire all grades and both sexes in all intervention groups. This led to the construction of the following seven groups: first-grade male, first-grade female, second-grade male, second-grade female, third-grade male, third-grade female, and others. Others were classified as participants where grade and/or sex was missing. After being categorised in this way, the individuals in each of the seven groups were computer randomised into three intervention groups (A–C) by a statistician, i.e. each individual was assigned the letter A, B or C. The three treatment groups were iMBI, Internet-based music therapy (iMT), and waiting list. The initial seven groups were only created for randomisation purposes. Of the 283 participants who gave written consent, 202 students—142 female, 50 male, and 10 where the information on sex and/or school was missing—also answered the Web-based questionnaires. The more rural school provided 45 students and the remaining 147 participants came from the more urban school. The mean age was 16.9 years and the median age 17 years (range 15–19 years). Both intervention groups represented Internet-based self-help programmes and the coordinator at the institution handled all contact with the study subjects. This was to preserve the single-blindness of the intervention to the researchers. The participants were allowed by the schools to do a 10-min daily intervention during lesson time and were asked to do at least one intervention on each school day. There were no reminders given to the students, i.e. they had to participate in their appointed intervention on their own incentive. The 8-week interventions commenced on March 15, 2012. The participants could, via e-mail, contact two of the co-authors of this study (CA or FT), who are also physicians, if they felt that their psychiatric health deteriorated during the interventions. Power was calculated for the main outcome of the study, improvement in global severity index (GSI) (see below) with the conventional values for significance level of 0.05 (α), and for power of 0.80 (β). With three groups to be compared, we used a one-way ANOVA pairwise two-sided equality calculation. We estimated a priori that with a population sample of 73 in each group, the GSI in the iMBI group would differ significantly from the waiting list group. This was based on a face-to-face mindfulness study in adults where GSI, our primary outcome, was measured [44]. In that study, the authors found an improvement in GSI from 0.91 (SD 0.71) to 0.53 (SD 0.51) and a median change of –43.2% after an 8-week MBSR course. According to the power calculation, we needed at least 219 participants, i.e. 73 in each group. To compensate for a presumed high pre-baseline dropout rate, we estimated the dropout rate before the first questionnaire to be 70%, based on two Swedish studies using GSI as an outcome in adolescents [45, 46]. We also made an estimation of the post-baseline dropout rate, of 44%, based on the responders that actually complied with an intervention in an adult iMBI study [43]. These assumptions led to an estimated total sample size of 1140 in the present study. We judged that the population of 1403 in the two chosen schools was adequate to address the study aims. A recent systematic review of 36 studies [47] listed the different definitions/measures of dropout and engagement. As it does not seem as if a consensus has been reached on what terminology is most appropriate, we discriminated those who logged in with those who did not as follows: ‘logged in’ and ‘not logged in’. We believe that this represents the most accurate description of those students who enrolled in our study [47, 48]. BODY.INTERVENTIONS: The two active interventions were Internet-based self-help programmes that were completely computerised without any human interference. They were based on modules of approximately 10 min and contained both video and audio material, but the focus was on the latter. Both interventions logged compliance in terms of log in identification, time at log in, and fulfilment of the session. They were accessible by any device that connects to the Internet, such as computers, smartphones, and tablets. All groups, including the waiting list group, got access to both interventions after all post-intervention questionnaires were completed. No further follow-up was performed. BODY.MINDFULNESS-BASED INTERVENTION (IMBI): The iMBI (In Swedish: Mindfulness Grundkurs 2.0 from Mindfulnesscenter AB, Sweden) was designed by Dr. Ola Schenström, a family physician and renowned national expert in Sweden on clinical mindfulness meditation. The programme is an 8-week course consisting of sessions of 10 min of mindfulness meditation twice daily, 6 days a week. The modules consist of standard mindfulness meditation techniques, such as body scan and mindfulness of breath, and other perceptions [49], and could be defined as an intervention based on mindfulness training [31]. The intervention incorporates elements from both MBSR [50] and more cognitively oriented parts from MBCT [51]. An iMBI, in a study on anxiety in adults, has used the same platform and guided meditations, but with cognitive material focused on anxiety [40]. For our study, a complete intervention was defined as at least 40 sessions, which is lower than the original programme but was deemed appropriate by the constructor of the programme. BODY.MUSIC THERAPY INTERVENTION (IMT): We chose iMT as the active control to iMBI, as one meta-analysis on music therapy showed a good effect in stress reduction from listening to music both in itself and in combination with music-assisted relaxation techniques, and concluded that the best effect is on adolescents [52]. Listening to music is also something that adolescents are generally interested in and tend to do, of their own free will, when feeling stressed or emotionally challenged [53]. Furthermore, a small study on adolescents targeting depression with music therapy, which included active participation with instruments and interpretation in terms of painting, showed promising results [53]. In addition, the partial similarity between the two Internet-based self-help programmes makes these two groups comparable. For the present study, a complete intervention was defined as at least 40 sessions. The iMT, Musikintervention, was designed with the aid of Professor Björn Ejdemo, MD, and visiting professor of music at the Australian National University, who made a preliminary selection of pieces of music. Per Vegfors, MD, specialist in child and adolescent psychiatry, assessed the appropriateness of the music from an adolescent psychiatric perspective. A total of ten non-vocal classical music pieces accessible on YouTube were chosen that met the criteria of (1) accessibility, i.e. being relatively easy to listen to for an untrained ear, (2) being of approximately 10 min in duration, and (3) recognisable as being calming or soothing. The programme Musikintervention (Paxx Media AB, Sweden) used streamed music videos from YouTube. See appendix for list of music and interpretations. BODY.QUESTIONNAIRES: We used three well-defined, well-validated, and reliable psychometric tests, before and after the intervention, that had previously been used on adolescents: the Symptoms Checklist 90 (SCL-90) [54], the Perceived Stress Scale (PSS-14) [55], and the Pittsburgh Sleep Quality Index (PSQI) [56]. The combination of scales was chosen to give an insight into the students’ perceived stress (PSS-14) and likely outcomes of that stress, expressed as low-quality sleep (PSQI) and increased general psychiatric symptoms (SCL-90), as previous studies have shown bi-directionally stress-associated impairments in psychiatric symptoms and sleep [11]. The time required to fill in the questionnaires was adjusted to avoid questionnaire fatigue and keep good test–retest reliability. BODY.SYMPTOMS CHECKLIST 90 (SCL-90): To measure general psychiatric symptoms, we used the 90-item SCL-90, which uses a five-point Likert scale to assess overall psychiatric symptoms, including somatisation. The main outcome of the scale is the GSI, which is calculated as the total sum of the weights for each individual item divided by the total number of questions answered (with a minimum answer rate of 80%). The SCL-90 is commonly used in psychiatric evaluations and has a subscale measuring somatisation [54]. The SCL-90 has satisfactory internal consistency reliability [57, 58] and test–retest reliability over both a week [54] and 10 weeks [58]. The latter study also showed a good test–retest reliability coefficient for the GSI. The validity of the scale has been confirmed with regard to internal structure, factorial invariance, and convergent–discriminant validity [54, 57, 59, 60]. BODY.PERCEIVED STRESS SCALE (PSS-14): For measurement of perceived stress, we used the widely accepted 14-item PSS, constructed as a five-point Likert scale [55, 61]. The PSS only focuses on how the individual experiences the stressors and not on their magnitude. The PSS-14 scale has been shown to have good internal consistency and reliability (Cronbach’s alpha 0.75–0.89), and to have good test–retest reliability over 2 days to 4 weeks. It has been empirically validated in college students [62]. Measurements of perceived stress have higher ecological validity than physiological response parameters, self-report of psychiatric symptoms, and behavioural changes and stressors, such as major life changes [63]. BODY.PITTSBURGH SLEEP QUALITY INDEX (PSQI): We used the PSQI [56] to measure sleep quality. This index is an algorithm that calculates sleep quality based on nine parameters (one of which is divided into eight sub-items) and results in a numerical value with a cutoff level for low-quality sleep of more than five points. The PSQI has been shown to have good test–retest reliability for both the global score and the subscores in both short-term (2 days) and long-term (1–2 months) follow-ups, with an overall Cronbach’s alpha of 0.87. The PSQI has also shown good correlations with sleep logs and lower, but significant, correlations with polysomnography, confirming its validity [64]. The tests were sent via e-mail to the address given by each student when signing the informed consent document. The students were able to answer the tests when it suited them during a window of ten consecutive days. All questionnaires were handled by the Internet-based survey programme Inquisite Survey System (Inquisite Inc., Copenhagen, Denmark). Data were stored unidentified to guarantee privacy and blinding. BODY.STATISTICAL ANALYSIS: To examine aim 1, i.e. to investigate whether there was a potential effect of the Internet-based self-help programmes on psychiatric and stress-related symptoms assessed before and after the intervention, we aimed to compare the scores of the different scales before and after the intervention among the active participants. We also used Student’s t test to investigate whether the three groups differed at baseline. These comparisons were performed to check whether the randomisation procedure was successful. To examine aim 2, i.e. to investigate the feasibility in terms of compliance to the interventions with the two Internet-based self-help programmes, we used a CONSORT flowchart to display the dropout rates at the different steps of the study [65]. The comparison in dropout rate between the two intervention groups (iMBI and iMT) was done using Fisher’s two-tailed exact test calculated with the number of participants that were randomised to either intervention and the participants that finished at least one session of either intervention. The Fisher’s two-tailed exact test was used as we intended to analyse whether the relatively small samples deviated from the null hypothesis that there was a difference between the dropout rates between iMBI and iMT. To examine aim 3, i.e. to investigate whether there is any association between compliance to the intervention and psychiatric and stress-related symptoms at baseline, we compared the participants that logged in and completed at least one session and those who did not log into the self-help programme. We compared whether there was a difference in psychiatric and stress-related symptoms between those two groups, separately in each intervention group (iMBI and iMT). We used a scatter plot to check whether the distribution was skewed and used the non-parametric Wilcoxon rank-sum test if the distribution was skewed and the parametric Student’s t test when the data were normally distributed. Statistical analysis was done using Stata IC 12 (Stata Corp, Texas, USA). BODY.RESULTS: 283 students were randomised into the three groups of the study: iMBI (n = 95), iMT (n = 94), or waiting list (n = 94); 1119 eligible candidates declined participation and 1 was excluded due to the language exclusion criterion. The randomisation worked well, as no significant differences were found between the three groups before the intervention in any of the scales. Of the 283 participants, 1 logged into both interventions and was excluded from the analysis. Of the 282 remaining students, 15 (15.8%) logged into the mindfulness intervention and 19 (20.4%) to the music intervention. Of the 19 persons who logged into the iMT, 4 did not complete any session, which was defined as playing at least 50% of the pieces of music. All but one person logging into the iMBI fulfilled at least one session. The flowchart is shown in Fig. 1. The distribution curve of sessions is shown in the supplementary Figure.Fig. 1CONSORT flowchart of the study in terms of compliance to the interventions. The participant who logged into both interventions was excluded after the randomisation. The percentages in the intervention groups are the proportions of the randomised participants assigned to each intervention. Interventions: iMBI Internet-based mindfulness-based intervention, iMT Internet-based music therapy, and WL waiting list Table 1 shows the comparisons between those who logged in and completed at least one session and those who did not log into the self-help programmes. No significant difference in any of the scales was found between those who logged in and completed at least one session and those who did not log into the self-help programmes.Table 1Differences between those who logged in to at least one session and those who did not log into the self-help programmes (i.e. iMBI Internet-based mindfulness-based intervention and iMT Internet-based musical therapy) in general psychiatric symptoms (GSI from Symptoms Checklist-90), perceived stress (PSS Perceived Stress Scale-14), and sleep quality (PSQI Pittsburgh Sleep Quality Index) ScaleIMBIIMTLogged inNot logged in p Logged inNot logged in p GSI n 13 63 15 56 GSI mean (SD) 0.96 (0.46) 0.84 (0.57) 0.78 (0.53) 0.83 (0.47) GSI CI 0.68–1.24 0.70–0.98 0.30a 0.48–1.07 0.71–0.96 0.59a PSS n 15 56 14 56 PSS mean (SD) 29.4 (7.43) 30 (8.52) 0.41b 30.7 (9.49) 28.7 (6.96) 0.82b PSS CI 25.3–33.5 27.7–32.3 25.2–36.2 26.8–30.5 PSQI n 14 57 13 55 PSQI mean (SD) 5.93 (0.89) 6.93 (0.42) 6.69 (3.20) 6.09 (2.53) PSQI CI 4.0–7.86 6.09–7.77 0.40a 4.76–8.62 5.41–6.77 0.65a a Skewed distribution—Wilcoxon rank sum b Normal distribution—Student’s t test Fisher’s exact test (two-tailed) gave a p value of 0.842 on comparing the number of participants who did at least one session of either intervention (iMBI or iMT), excluding the person who logged into both interventions. The data below are only descriptive, since the compliance rate to the interventions was low. The compliance to the study protocol was 72% at baseline. The only participant who did a complete intervention, defined as at least 40 sessions, performed 42 iMBI sessions and decreased the GSI by 31% (from 0.80 to 0.55). The same person decreased in PSQI from 8 to 7 and in PSS from 29 to 23. As regards the participants completing at least ten sessions (i.e. 25%), there were four in the iMBI group, three of whom answered the post-intervention questionnaires. In the iMT group, only one participant completed at least 25% of the sessions. The iMBI group decreased their mean GSI from 0.85 to 0.63 (SD 0.13 and 0.19, respectively). Their PSS decreased from 29.0 to 26.3 (SD 6.0 and 5.8, respectively). PSQI decreased from 9 to 8 (SD 4.6 and 5.5, respectively). The only iMT participant fared worse, with GSI increasing from 0.68 to 0.90, PSQI increasing from 6 to 7, but with PSS decreasing from 26 to 24. None of the participants used the possibility to contact a physician via e-mail. BODY.DISCUSSION: Our main finding in this RCT is that the compliance rates of the two Internet-based self-help programmes were very low in adolescents (aim 2). This in itself is of interest, although the limited compliance did not allow us to investigate our first aim due to insufficient statistical power. However, the study provides useful information for the planning of future studies. We found no association between psychiatric and stress-related symptoms and compliance in any of the intervention groups (aim 3). Our second aim, to determine feasibility in terms of compliance to the intervention, shows that the feasibility in conducting Internet-based self-help programmes in mindfulness may be limited in adolescents and that more research is needed on why the compliance is low in non-clinical samples. A limited compliance to an intervention may occur in several steps and present as failure to engage in the intervention or drop out from the protocol. In the present study, the low compliance after the randomisation/baseline assessment mainly occurred in two steps. First, a limited number of the adolescents actually logged in: only 15 of the 95 and 19 of the 94 adolescents logged into the iMBI and iMT, respectively. Second, the adolescents who actually logged in only completed a limited number of sessions and only one adolescent completed all 40 sessions. The positive effects of mindfulness training are partly related to compliance rates. A recent meta-analysis on face-to-face MBIs in adolescents showed a positive correlation between minutes of mindfulness training and effect sizes [32]. Kuyken et al. reported that at least 50% attendance at the eight sessions of MBCT is considered necessary to receive an adequate treatment dose [66]. Thus, more knowledge is needed on the potential causes behind low compliance as well as methods to increase compliance with iMBI. BODY.POTENTIAL CAUSES BEHIND AND WAYS TO OVERCOME LOW COMPLIANCE RATES: According to Christensen et al. [67], there are three general approaches to investigate compliance in Internet-based interventions for anxiety and depression; these approaches can also be used in other types of patients and interventions. The first involves the examination of correlations to personality, demographic, and service delivery factors. The second includes the use of post-test qualitative investigations to obtain retrospective analyses of peoples’ perceptions of trial participation and barriers to the use of the Internet intervention, and the third involves experimental manipulation of variables thought to change compliance. The first approach has shown that lower compliance is associated with higher levels of emotional distress [67], certain types of conditions (higher compliance in chronic headache patients than in weight control patients), and male sex combined with lower GSI in cardiac patients [68]. Kabat-Zinn and Chapman-Waldrop found, however, no association between compliance and GSI in referred somatic and psychiatric patients [68]. Compliance rates may decrease in Internet-based [18, 69], longer, and preventive (rather than treatment) interventions in healthy population-based samples [70]. We found no evidence of an association between compliance rates and psychiatric and stress-related symptoms in our study population of non-clinical Swedish adolescents, which is in accordance with the study by Kabat-Zinn and Chapman-Waldrop who concluded that “there was no indication that severity of symptoms or illness affected outcome in terms of programme completion”. It is possible that feelings of shame or embarrassment could have been behind our students’ reluctance to log into the interventions during class time, although it also was possible for the students to log in outside class time. Another possibility is that some students may have found the intervention to be anxiety inducing, as previous studies have shown that emotional distress [67] decreases compliance. The second and third approaches for compliance investigation according to Christensen et al. were not included as part of our study. Anecdotally, some students found the intervention ‘boring’ and others said that they did not complete the intervention due to lack of time. A teacher, school nurse, and school counsellor were of the opinion that stress, due to school issues, made the students less compliant to such interventions. Both students and personnel mentioned that stress interventions should be part of the curriculum to increase compliance and decrease stress. In an RCT conducted in Belgian schools, where mindfulness classes were part of the curriculum, the compliance rate was 85% [71]. Previous studies have examined how compliance to iPTs can be increased. For example, an Australian study found that compliance to iCBT in adults could be increased with e-mail reminders [69]. Some studies have found that guidance by trained psychotherapists augments the participation rate [72], while other studies, including university students, have found no such effect [73]. Material incentives have been shown to enhance compliance in children and adolescents as well as adults [74–77]. These potential causes and potential ways to increase compliance (see above) may be more or less salient during the different phases of the intervention, i.e. there may be different types of resistance towards entering and staying in the programme or, in this case, to log in or not log into the Internet-based self-help programme. For example, embarrassment and shame, gender, or a wish to belong to a specific intervention may deter students from logging in, whereas at later stages in the study other factors may lead to dropouts, such as psychological distress/anxiety, programme content, and lack of incentives. Some factors may lead to low compliance during the entire intervention, such as lack of time and stress, which may call for the inclusion of stress interventions into the curriculum. BODY.LIMITATIONS AND STRENGTHS: There are several limitations with the present study. We had no strategy to address any low compliance in the present study apart from our attempts to increase the sample size. However, an important aim was to study the potential feasibility of the two Internet-based self-help programmes in an RCT that targeted students in their adolescence, which represents a novel contribution. The low compliance rate did not allow for sufficient power to investigate our primary aim regarding the potential effect of the two interventions. In addition, we have only anecdotal information on why the adolescents dropped out. The use of questionnaires could have led to self-report bias, but questionnaires have been found to provide reliable data in Nordic adolescents [78]. Another limitation of this intervention is that it might be dubious to introduce stress management methods, such as mindfulness, with a sole focus on the individual while neglecting the overall organisation and structures in the school setting, which in itself may entail stress-inducing characteristics. Instead, a relational approach, which sees mindfulness as a socially contingent resource for communities, has been suggested as an alternative to the more common focus on the individual [79, 80]. Finally, although not being a direct limitation of the study, one could argue that all types of school interventions might compete with other important activities, such as schoolwork. However, there are also several strengths that could be useful in future studies. For example, we included an active control group, listening to music, which is something that adolescents tend to do when feeling stressed or emotionally challenged [53]. A focus on listening is sometimes included in MBI, as in MBCT [51], and the partial similarity between the two interventions [81] makes these two groups comparable in terms of focus, although the focus in mindfulness is purposeful and without judgment. Another strength is that we evaluated the compliance in several steps [67]. The study design included reliable, well-validated questionnaires, and no adverse reactions to any of the interventions were reported. Finally, this is the first RCT that aimed to compare two different Internet-based self-help programmes in both male and female adolescents. BODY.CONCLUSIONS: In conclusion, the compliance rates of the two Internet-based self-help programmes were very low in adolescents, which did not allow us to investigate our primary aim. The study provides, however, useful information for the planning of future studies in adolescents. Future studies need to examine potential causes behind why compliance rates may be low in Internet-based self-help programmes and how compliance rates can be increased in adolescents, as the potentially positive effects of mindfulness programmes on psychiatric symptoms and stress most likely are related to compliance rates. BODY.ELECTRONIC SUPPLEMENTARY MATERIAL: Below is the link to the electronic supplementary material. Supplementary material 1 (DOCX 98 kb) Supplementary material 2 (DOCX 81 kb)

TITLE: Treatment of Danhong Injection Combined with Naoxintong Capsule in Acute Coronary Syndrome Patients Undergoing PCI Operation: Study for a Randomized Controlled and Double-Blind Trial ABSTRACT.OBJECTIVE: This double-blind and randomized placebo-controlled trial evaluated the safety and efficacy of Danhong injection combined with Naoxintong capsule in patients with acute coronary syndrome (ACS) after percutaneous coronary intervention (PCI). ABSTRACT. METHODS: ACS patients scheduled to undergo PCI (n = 130) were equally and randomly apportioned to either a treatment or control group. After PCI, the treatment group received Danhong injection combined with Naoxintong capsule for 12 weeks, while the control group was given placebo. Both groups were otherwise treated with conventional secondary prevention of coronary artery disease. The groups were primarily evaluated for clinical efficacy and cardiovascular events. Evaluative indicators of safety included adverse events, platelet count, and liver, renal, and blood coagulation functions. ABSTRACT. RESULT: No cardiovascular events or adverse reactions were observed in either group. The treatment group demonstrated better signs of clinical efficacy, including left ventricular ejection fraction, higher nitric oxide levels, and lower levels of endothelin-1 (ET-1) and von Willebrand factor (VWF). ABSTRACT. CONCLUSION: ACS patients treated with Danhong injection combined with Naoxintong capsule after PCI demonstrated better improvement with regard to markers associated with atherosclerosis and adverse cardiovascular events, without apparent adverse effects. Thus, Danhong injection combined with Naoxintong capsule was safe and effective for treating ACS patients after PCI. BODY.1. INTRODUCTION: Acute coronary syndrome (ACS) generally refers to a sudden decrease in blood flow to the coronary arteries, or acute myocardial ischemia, including ST segment elevation myocardial infarction (STEMI), non-STEMI (NSTEMI), and unstable angina. ACS is a worldwide health issue with high morbidity and mortality [1] and is the second leading cause of death in coronary heart disease. In China, rates of hospitalization and hospital mortality due to ACS have been rising and the disease is now a leading cause of death and disability [2]. The mainstay treatment for ACS is percutaneous coronary intervention (PCI), which can quickly reconstitute the blood supply and effectively remove vascular stenosis or occlusion [3]. However, PCI cannot prevent the progression of atherosclerosis. Rather, PCI has the potential to promote the formation of early acute or subacute small blood clots and activate platelets, subsequently leading to stent restenosis [4]. Indeed, it is widely believed that vascular endothelial dysfunction is the initiating factor for atherosclerosis [5], and stent restenosis has been linked to injury of vascular endothelial cells in the coronary artery intima during PCI [6]. In addition, PCI-induced vascular endothelial cell dysfunction was found to promote plaque rupture and thrombosis, leading to the conclusion that this was the main cause of stent restenosis after PCI. Markers of endothelial function include plasma nitric oxide (NO), endothelin-1 (ET-1), and von Willebrand factor (VWF). NO is an indicator of vasodilatation [7], and ET-1 is a vasoconstrictor [8]. Elevated plasma levels of VWF have been linked to thrombosis [9]. Abnormal levels of NO, ET-1, and VWF promote endothelial dysfunction that has been associated with atherosclerosis and as such are important indicators in vascular biology and pathophysiology. Indeed, levels of circulating NO, ET-1, and VWF are independent predictors of coronary thrombosis, stent restenosis, and other adverse cardiovascular events [10]. Danhong injection is a traditional Chinese medicine (TCM) that promotes blood circulation to remove blood stasis and regulate body function [11]. Its main components are Tanshinone and Salvia miltiorrhiza safflower yellow pigment, Salvia miltiorrhiza phenolic acid, safflower phenolic glycosides, and catechol. Studies have shown that Danhong injection can prevent atherosclerosis, ameliorate oxidative stress, attenuate inflammatory injury, regulate blood lipids, balance blood pressure, and reduce blood viscosity [12]. In addition, Danhong injection was shown to significantly reduce myocardial cell degeneration and necrosis caused by ischemia, alleviate the onset of angina pectoris, and improve myocardial blood perfusion [12]. The major components of Naoxintong capsule are milkvetch root, leech, earthworm, scorpion, Salvia miltiorrhiza, Angelica sinensis, Radix Paeoniae Rubra, Rhizoma Ligustici wallichii, peach kernel, safflower, frankincense, myrrh, Caulis Spatholobi, ramulus mori, achyranthes, and cassia twig. Naoxintong capsule supplements qi, that is, the basic material that makes up the human body and blood circulation according to the theory of TCM [13]. Previous studies reported that Naoxintong capsule protected myocardial and endothelial cells, promoted coronary collateral circulation [14], and reduced the risk of cardiovascular disease morbidity. Danhong injection and Naoxintong capsule have been widely used in a variety of cardiovascular and cerebrovascular diseases. However, no randomized controlled trials have been performed to evaluate the safety and efficacy of the combined use of Danhong injection and Naoxintong capsule for treatment of ACS patients after PCI. The present study was a multicenter, randomized, double-blind, placebo-controlled, and parallel design clinical trial that investigated the safety and efficacy of the combined use of Danhong injection and Naoxintong capsule in patients with ACS after PCI. We explored the molecular basis underlying the beneficial effects of the combined use of these two Chinese traditional medicines in these patients. BODY.1.1. ETHICS: The Ethics Committee at Guangdong Hospital of Traditional Chinese Medicine reviewed and approved the study. All patients signed informed consent before the study, and the study was conducted in accordance with the principles of clinical practice and Helsinki declaration. The trial was registered in the China Clinical Trial Registration Center (registration number: ChiCTR-IOR-14005693). The test protocol was approved by the Ethics Committee of Guangdong Provincial Hospital of Traditional Chinese Medicine (ethical batch number: B2014-031-01). BODY.2. METHODS: BODY.2.1. TRIAL DESIGN: This study was a multicentered, randomized, double-blind, placebo-controlled, and parallel-designed clinical trial involving ACS patients after PCI. All patients who met the inclusion criteria were randomly allocated to the control group and the treatment group according to the ratio of 1 : 1 based on the random numbers that were computer-generated by researchers who did not work for the research institutions (i.e., the clinical research methodology team at Guangdong Hospital of TCM). The numbers were the same as those shown on the medication package and the drug package used in the study. The study was double blinded; that is, patients and medical staff were blinded to the treatment and placebo groups, but not the data managers. Only the data manager in each center knew which group a patient was assigned to. BODY.2.2. PARTICIPANTS: ACS patients scheduled to undergo PCI (n = 130) from September 2014 to December 2015 were selected for this study. Eighty of the patients were treated at Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Province), and 50 were at Maoming City People's Hospital. Patients were apportioned to a treatment group or a control group according to a random number table (Figure 1). BODY.2.2.1. INCLUSION AND EXCLUSION CRITERIA: Patients who met the following conditions were included in this study: a diagnosis of ACS (see below); age: 50–80 y; and informed consent signed by self or relatives. Patients with any of the following were excluded from our study: allergy to Danhong injection or Naoxintong capsule; no inspection record to evaluate safety and efficacy indicators; or use of any drug during the study that would affect the evaluation of clinical effects or safety. In addition, patients with any of the following diseases or complications were excluded: serious cardiac arrhythmias (e.g., type II atrioventricular block or sick sinus syndrome); severe hypotension; hypertension emergency; severe heart failure (cardiac function > III not effectively controlled); acute cardiac tamponade; acute pulmonary edema; hemorrhagic cerebrovascular diseases; gastrointestinal hemorrhage; hemophilia; severe thrombocytopenia; coagulation disorder; serious diabetic complications (diabetic ketoacidosis); severe liver or kidney impairment (serum alanine aminotransferase [ALT] > 3-fold normal ceiling or serum creatinine ≥ 265 μmol/L); malignant tumor; psychosis; or coronary artery bypass grafting surgery (i.e., heart bypass surgery). Participants were considered lost during the study if the researchers determined that the subject should discontinue due to aspartic acid transaminase (AST) or ALT > 3-fold the upper limit of the normal range in 2 consecutive examinations; creatinine > 1.5-fold the upper limit of normal in 2 consecutive examinations; severe adverse reactions; other diseases which influenced the curative effects or evaluation of adverse events; the participant being not suitable for continuing use of the study drug; or the patient becoming unblinded to the treatment. In addition, patients were dropped from the study if they discontinued participation by withdrawing informed consent, were unwilling to continue because of poor effects or symptomatic deterioration, could not tolerate the adverse events, or died. ACS was diagnosed based on the recommended criteria of the American College of Cardiology/American Heart Association with regard to STEMI and NSTEMI-ACS. Characteristics of STEMI included symptoms associated with myocardial ischemia, ST segment elevation on electrocardiogram (ECG), and a change in myocardial damage markers (troponin I, troponin T, and creatine kinase isoenzyme). Blood samples for the detection of myocardial damage markers were collected ≥ 2x within an interval of 6 hours. Features of NSTEMI-ACS were apparent within one month of new angina, or original angina progression within one month or resting state angina, accompanied by a deelevation of the ST segment on ECG. NSTEMI was diagnosed if the concentrations of the myocardial damage biomarkers (troponin I, troponin T, and creatine kinase isoenzyme) were above the normal range. Otherwise unstable angina was considered if the myocardial damage biomarkers were within normal. BODY.2.2.2. SAMPLE SIZE DETERMINATION: According to [15], the effective rate of secondary prevention of coronary heart disease in patients with ACS in China is 76%, and the effective rate of secondary prevention of coronary heart disease combined with Danhong injection is 86%. Assuming δ = 0.08, α = 0.05, and β = 0.20 and using a noninferiority test design (i.e., the treatment group after 3 months has a higher efficiency than the control group), the following formula was used to determine the sample size: (1)n1=n2=Z1−α/2+Z1−β2π11−π1+π21−π2π1−π2−δ2.Thus, the number of samples should be at least 54 in each group. Considering a 20% loss rate, it was estimated that there should be a minimum of 65 cases in each group and at least 130 cases overall. BODY.2.2.3. RANDOMIZATION: A hierarchical stochastic allocation scheme was used for randomization, in which the stratification factor was 2 centers. Each center adopted the method of block randomization to set the block size to 4, and the patients who met the inclusion criteria were randomly allocated to the control group and the treatment group according to a 1 : 1 ratio. A randomized, multicenter trial was performed by nonparticipating research institute researchers (from the Clinical Research Methodology Group of Chinese Medicine and Guangdong Provincial Hospital of Traditional Chinese Medicine). The numbers were the same as those shown on the medication package and the drug package used in the study. BODY.2.3. INTERVENTION: Danhong injection (specification: 10 mL/branch; drug batch number: 14081017) and Naoxintong capsule (drug batch number 141080) were provided by Shaanxi Buchang Pharmaceutical. The placebo injection consisted of 0.9% sodium chloride. The Danhong injection and placebo injections were blinded by shading the intravenous infusion. The placebo capsules to replace Naoxintong capsules in the control group contained excipients and were provided by Shaanxi Buchang Pharmaceutical. The Naoxintong capsules and placebo capsules were identical in appearance, weight, packaging, specifications, and fonts. BODY.2.4. DRUG DELIVERY METHODS: All patients who met the inclusion criteria were given aspirin 300 mg, Plavix 300 mg, and atorvastatin calcium tablet 40 mg (chewing) after admission. For the patients with STEMI, PCI after thrombolysis or selective PCI was chosen according to the time interval from the onset of chest pain to admission and hemodynamic factors. For the unstable angina/NSTEMI patients, high-risk patients underwent PCI treatment as soon as possible, while low-risk patients had elective PCI therapy. Eventually, all patients who underwent PCI surgery were given secondary prevention of coronary artery disease after surgery, including aspirin (100 mg/d), Plavix (75 mg/d), atorvastatin calcium tablet (20 mg/d), angiotensin converting enzyme inhibitors (ACEIs), angiotensin II receptor antagonists (ARBs), and low molecular heparin. Those patients with no contraindications were given β-adrenergic-receptor blockers. Patients in the treatment group were given Danhong intravenous injection (20 mL Danhong added in 5% glucose 100 mL/0.9% sodium chloride, 100 mL, gtt, qd, 1x/wk) for one week and then Naoxintong capsule (4x, po, tid, 11 wk) for 11 weeks. The control group was given a placebo injection and placebo capsules as follows: placebo injection, 20 mL sodium chloride injection added in 5% glucose injection 100 mL/0.9% sodium chloride injection 100 mL iv, gtt, qd × 1 week; and placebo capsule, starch preparation, 4/time, po, tid × 11 week. The total treatment lasted 12 weeks. The end point or an early termination point of the trail was considered the completion of observation, including the baseline and double-blinded treatment period of 1 week and 12 weeks for a total of 3 times. Patients who discontinued were considered lost to the study. The following were recorded upon hospitalization on the first day, the first week, and the second week: number of chest pains, routine blood examination, liver function, renal function, blood coagulation, ECG, levels of NO, ET-1, VWF, adverse reactions, and cardiovascular events. BODY.2.5. OUTCOMES: The primary outcomes were clinical curative effects and cardiovascular events. Clinical curative effects were rated excellent, effective, or ineffective, based on the clinical efficacy criteria recommended by the Drug Administration of the National Ministry of Health. A curative effect was considered excellent when symptoms disappeared or improved significantly, the frequency of chest pain attacks was reduced by 80%, and ECG returned to normal or roughly normal. Results of treatment were defined as effective if symptoms improved; chest pain attacks were reduced by 50–80%; ECG showed a decreased ST segment ≥ 0.05 mV, ST segment fell ≥ 0.05 mV but did not return to normal, T wave in the primary lead changed from flat to upright, or >50% of the T wave inversion was lighter. The treatment was considered ineffective when either there was no change in clinical symptoms or symptoms aggravated; chest pain attacks showed no significant change; and the ST segment on ECG showed no change or aggravated. Cardiovascular events within 3 months were recorded as follows: nonfatal myocardial infarction; vascular blood supply and revascularization; hospitalization because of severe angina or heart failure; or resulting in mortality. Adverse cardiovascular events included cardiac death, cardiac shock, arrhythmia, repeat myocardial infarction, revascularization, readmission due to ACS, recurrent myocardial ischemia, cardiac insufficiency, or nonfatal stroke. In addition, general adverse events or reactions were observed. Since this study used listed drugs, the adverse events that occurred during this trial were mainly dizziness, anxiety, and stomach pain. Adverse events or reactions, as well as new disease or aggravation of original symptoms, were recorded. Serious adverse events were reported according to the requirements of good clinical practice. The secondary outcomes included parameters from laboratory examinations: plasma ET-1, NO, VWF, and cardiac function such as left ventricular ejection fraction (LVEF) through echocardiography. Safety observation indexes included routine blood exam and liver, renal, and coagulation functions. Regular laboratory routine tests included blood, urine, stool, liver and renal functions, 4 items of blood lipids, and fasting blood-glucose. For the endothelial function index, 6 mL of blood was taken from the elbow vein of all patients (fasting) and centrifuged. The separated plasma was immediately frozen at −450°C. The levels of circulating ET-1, NO, and VWF were measured at the King Med Diagnostics Center. BODY.2.6. STATISTICAL ANALYSIS: According to the intention-to-treatment population analysis principle, the last observation carried forward method was used for analysis of clinical efficacy to deal with missing data and information dropout. Baseline data of the study were computed by corresponding appropriate statistical analyses based on the nature of the data, that is, the mean for continuous variables, and frequency distribution for classification variables. Clinical efficacy was analyzed by comparing changes in the evaluation indexes in the respective individuals of the 2 groups from baseline to each observation period. All analyses were performed using a 2-sided test, and P < 0.05 was considered statistically significant. Measurement data are described as the number of cases, mean ± standard deviation, minimum, maximum, and the median and were statistically analyzed using a t-test, analysis of variance, or Wilcoxon rank sum test. Various count data were described as case number and percentage and were statistically analyzed using the chi-squared or Wilcoxon rank sum tests. Ranked data are described as the number of cases and percentage and statistically analyzed using the Wilcoxon rank sum test. BODY.3. RESULTS: BODY.3.1. DEMOGRAPHIC AND BASAL CLINICAL CHARACTERISTICS: A total of 130 ACS patients, all of whom had either NSTEMI or STEMI and were scheduled for PCI, were enrolled in the present study (Table 1; Figure 1). The 130 patients were equally and randomly assigned to receive either treatment consisting of Danhong injection and Naoxintong capsule (treatment group; 36 men and 29 women; aged 68.88 ± 5.75 years) or a placebo injection and placebo capsule (control group; 38 men and 27 women; aged 67.26 ± 5.49 years). There were no statistically significant differences between the treatment and control groups with regard to the following demographic and basic clinical characteristics: age, gender ratio, blood pressure, body mass index, distribution of risk factors of coronary heart disease, and cardiac function classification according to the New York Heart Academy. BODY.3.2. CLINICAL EFFICACY: During a 12-week follow-up period, 6 and 3 cases were lost in the treatment and control groups, respectively, and these patients were removed based on intention-to-treat. In the twelfth week of follow-up, for the treatment group the clinical curative effects were judged as excellent, effective, and ineffective in 35, 16, and 8 patients, respectively (Table 2). In the control group, the curative effects were rated excellent, effective, and ineffective in 27, 17, and 18 patients. The overall response rate of the treatment group (86.44%) was significantly higher than that of the control group (70.96%). BODY.3.3. CARDIOVASCULAR EVENTS: During the 12-week treatment period, no patient in either group experienced any serious event such as death, nonfatal myocardial infarction, target blood supply vessel revascularization, recurrence of myocardial infarction, hospitalization for recurrent myocardial ischemia or severe heart failure, or severe angina events. BODY.3.4. SECONDARY INDEXES: BODY.3.4.1. CIRCULATING NO: Prior to treatment, the mean levels of NO of the treatment and control groups were similar (P > 0.05; Table 3). At 7 days of treatment the levels of NO had increased in both groups, but the change in the treatment group was significantly higher than that of the control group. At 12 weeks of treatment the level of NO had significantly elevated in both groups, but the change in the treatment group was obviously larger than that in the control group (P < 0.05). Therefore, we conclude that combined use of Danhong injection and Naoxintong capsule substantially increases the circulating level of NO in ACS patients undergoing PCI compared with the control group. BODY.3.4.2. CIRCULATING ET-1 AND VWF: Prior to treatment, the mean levels of ET-1 and VWF of the treatment and control groups were similar (P > 0.05; Table 3). At 7 days of treatment, the levels of ET-1 and VWF had decreased in both groups, but the change in the treatment group from baseline was less than that of the control group. At 12 weeks of treatment, the ET-1 and VWF levels of both groups were significantly lower than at the baseline, but the decrease in the treatment group was significantly less than that of the control. Hence, we conclude that the combined use of Danhong injection and Naoxintong capsule sustains ET-1 and VWF levels better than the placebo in ACS patients after PCI. BODY.3.4.3. LVEF: Prior to treatment, the mean LVEF of the treatment and control groups were statistically similar (Table 4). At 7 days of treatment, the LVEF had increased in both groups, but the LVEF of the treatment group was significantly higher than that of the control group. It was surprising that, at 12 weeks of treatment, the LVEF of both groups were less than those at 7 weeks, but the LVEF remained significantly higher in the treatment group than the control group. Collectively, the combined use of Danhong injection and Naoxintong capsule improved cardiac function better than the placebo in ACS patients undergoing PCI. BODY.3.5. EVALUATION OF SAFETY: Based on the principle of intention-to-treat, the last observation carried forward approach was used to deal with missing data (Table 5). No significant differences in the levels of PLT, AST, ALT, BUN, CR, and INR were observed between the treatment and control groups at baseline, and after treatment these levels had not changed from baseline. This indicated that neither the combined use of Danhong injection and Naoxintong capsule nor secondary coronary heart disease prevention had any significant influence on the functions of major organs. BODY.3.6. ADVERSE REACTIONS: BODY.4. DISCUSSION: We employed a randomized, double-blind study design to explore the clinical benefits of the combined use of Danhong injection and Naoxintong capsule for ACS patients after PCI. Compared to the control patients, we found that the combination of these traditional Chinese medicines was associated with clinical benefits and improved endothelial function, without detectable cardiovascular events or other adverse reactions. ACS is a clinical syndrome caused by myocardial ischemia due to thrombosis. Myocardial reperfusion therapy is the most direct and effective treatment for STEMI and NSTEMI, and PCI is recognized as the mainstay means to achieve blood reperfusion in the shortest time [3]. According to China's PCI guide (2009) for treatment of unstable angina and NSTEMI, the selection of PCI therapy should be based on risk stratifications and is not recommended for patients who are at low-risk While there has been rapid progress in PCI technology for the treatment of ACS, some clinical issues after PCI remain, such as promoting the formation of early acute and subacute small blood clots, activating platelets leading to stent restenosis, promoting antiplatelet drug resistance, and leading to poor myocardial reperfusion [5]. Some clinical studies have suggested that the use of TCM alone, or TCM integrated with Western medicine, for the treatment of ACS can offer significant benefits to patients [11]. For example, Danhong injection was shown to promote blood circulation under conditions of blood stasis, regulate the body's physiology, and activate antiplatelet aggregation, anti-inflammation, antiapoptosis, and vascular endothelium protection [16]. Naoxintong capsule was shown to supplement qi, activate blood circulation, and eliminate hemostasis and free channels, and it also possessed antioxidant, antihyperlipidemic, antiatherosclerosis, and vasodilation properties [17]. Thus, Danhong injection and Naoxintong capsule have been widely used in a variety of cardiovascular and cerebrovascular diseases. In addition, we did not observe any associated adverse effects, although rare adverse reactions linked to Danhong injection have been reported [18]. Therefore, we believed that the combined use of these two medicines was safe and effective for treatment of ACS patients after PCI. Vascular endothelial cells, as the largest autocrine, paracrine, and endocrine organs in humans, regulate the dynamic balance of environment for blood vessel walls, both inside and outside, mainly by compounding and releasing the vascular active factor. NO and ET-1 are the two main vascular active factors, while NO influences vasodilatation, antiplatelet aggregation, antithrombosis, and inhibition of excessive proliferation of smooth muscle cells [8, 19]. VWF is a glycoprotein synthesized and secreted by vascular endothelial cells and macrophages that can cause blood vessels to contract [20]. Therefore, the balance between NO and ET-1/VWF is important for the homeostasis of vascular endothelial cells. An increase in ET-1/VWF and decrease in NO can cause vasospasm, smooth muscle cell proliferation, blood clots, and the hardening of plaques [21]. Hence, NO, ET-1, and VWF are considered sensitive indexes of endothelial cell injury and risk factors for thrombosis, severity of morbidity, and prognosis. In the present study, we found that Danhong injection combined with Naoxintong capsule was associated with significantly greater NO levels and lower plasma levels of ET-1 and VWF compared with the control group, which probably contributed to the benefits of these medications for ACS patients after PCI. Previous studies have shown that ACS is not completely dependent on the degree of coronary narrowness but rather is closely related to endothelial dysfunction [22, 23]. Vascular endothelial dysfunction can lead to a reduction in NO and promote platelet aggregation and infiltration of inflammatory factors, which predisposes an unstable plaque to rupture, subsequently leading to ACS [24, 25]. Therefore, although PCI is the mainstay treatment for ACS, it also causes clinical side effects including stent restenosis. Two major mechanisms contribute to PCI-linked restenosis. Firstly, PCI causes a decrease in NO release and increases in ET-1 and VWF [26, 27], promoting the release of angiotensin and stent restenosis. Secondly, PCI causes vascular endothelial cell injury, a subsequent decrease in anticoagulation substances, and the formation of subacute and late thrombosis [28]. Thus, vascular endothelial cell dysfunction is well recognized as the main mechanism leading to PCI-linked stent restenosis [29, 30]. Conversely, improving endothelial cell function was found to prevent stent restenosis after PCI [31]. In the present study, the combined use of Danhong injection and Naoxintong capsule ameliorated changes in the levels of circulating NO, ET-1, and VWF in our patients and, therefore, endothelial function as well. BODY.4.1. LIMITATIONS: Although we obtained positive results in the present study, we did not examine end points such as all-cause mortality, and a follow-up time of 3 months is relatively short. In addition, the sample size was relatively small although it reached requirement for power analysis. Thus, a multicenter study with a large cohort should be performed to corroborate our observations. BODY.5. CONCLUSION: Danhong applied as an injection is not appropriate for long-term use mainly due to the following: First, as an injection for intravenous infusion, the administration of Danhong is inconvenient for long-term use. Second, Danhong injection promotes blood circulation and is a collateral analgesic, which is only suitable for short-term use during the acute phase; long-term use will increase qi, which is not appropriate. ACS within 1 week is considered the acute phase, so Danhong injection should be used during this period. However, in the clinical setting Danhong injection is practical when combined with Naoxintong capsule. This conforms to the principle of Chinese medicine: to treat acute conditions symptomatically and chronic conditions radically. From the present study, we conclude that the combined application of Danhong injection and Naoxintong capsule can benefit ACS patients after PCI, compared with Western medications alone, by improving endothelial function. Furthermore, the treatment did not cause any detectable adverse reactions. Therefore, Danhong injection combined with Naoxintong capsule for treatment of ACS patients undergoing PCI is safe and effective.

TITLE: Bromfenac 0.09% bioavailability in aqueous humor, prophylactic effect on cystoid macular edema, and clinical signs of ocular inflammation after phacoemulsification in a Mexican population ABSTRACT.PURPOSE: The purpose of this study was to evaluate the aqueous humor bioavailability and clinical efficacy of bromfenac 0.09% vs nepafenac on the presence of cystoid macular edema (CME) after phacoemulsification. ABSTRACT.MATERIAL AND METHODS: A Phase II, double-blind, masked, active-controlled, multicenter, clinical trial of 139 subjects, randomized to either a bromfenac 0.09% ophthalmic solution (n=69) or nepafenac 0.1% (n=70). Subjects instilled a drop three times a day for a period of 30 days. Follow-up visits were on days 2, 7, 15, 30, and 60. Biomicroscopy, clinical ocular signs, and assessment of posterior segment were performed. The primary efficacy endpoints included the presence of CME evaluated by optical coherence tomography. Safety evaluation included intraocular pressure, transaminase enzymes, lissamine green, and fluorescein stain. ABSTRACT.RESULTS: The demographic and efficacy variables were similar between groups at baseline. The presence of pain, photophobia, conjunctival hyperemia, chemosis, cellularity, and corneal edema disappeared by day 30 in both groups. The central retinal thickness did not show significant changes after treatment when compared to baseline as follows: in the bromfenac group (247.2±32.9 vs 252.0±24.9 μm; P=0.958) and in nepafenac group (250.8±34 vs 264.0±34.1 μm; P=0.137), respectively. A statistically significant difference was observed between bromfenac and nepafenac group: (252.0±24.9 vs 264.0±34.1 μm; P=0.022), at day 30, respectively; even though there was no clinical relevance in the presentation of CME. There were no significant alterations in intraocular pressure, either lissamine green or fluorescein stains. The adverse events were not related to the interventions. ABSTRACT.CONCLUSION: Bromfenac 0.09% ophthalmic solution showed similar clinical efficacy to reduce the presentation of CME after phacoemulsification compared to nepafenac 0.01%. BODY.INTRODUCTION: Cystoid macular edema (CME) is a common cause of poor visual outcome after a complicated phacoemulsification. Usually CME is subclinical and its prevalence varies (5%–20%) depending on the diagnostic test.1–4 The pathophysiology involves an increase in vascular permeability due to prostaglandins’ elevated concentration as a consequence of a surgical inflammatory response by the rupture of cell membranes. However, the exact cause of CME is poorly understood.5 Management of inflammation is a mainstay after phacoemulsification.6 Non-steroidal anti-inflammatory drugs (NSAIDs) block the prostaglandin response through inhibiting COX enzymes.7 The use of NSAIDs has been particularly associated with adverse events (AEs) in the ocular surface.8–10 Bromfenac is an NSAID with bromine atom at the C4 of the benzoyl ring position. Its chemical structure lengthens the duration of anti-inflammatory activity and enhances the absorption into the ocular tissues.11–14 Nepafenac is converted by the ocular tissues to amfenac, which is an active metabolite that inhibits COX enzymes.15,16 Even though both of them, bromfenac and nepafenac, have been shown to be effective, clinical studies have not yet concluded which one is better.6 Notwithstanding the fact that bromfenac ophthalmic solution has been evaluated in numerous clinical studies in Japan, USA, and other countries, in Mexico bromfenac is not available commercially and consequently no clinical data have been collected. The purpose of the present study was to evaluate the aqueous humor bioavailability and clinical efficacy of bromfenac 0.09% vs nepafenac on the presentation of CME in Mexican patients after phacoemulsification. BODY.PATIENTS AND METHODS: BODY.STUDY DESIGN: A parallel, randomized, double-blind, active-controlled, multicenter clinical trial was designed to compare the efficacy of two ophthalmic solutions. The study was conducted at four research centers in Mexico. The sample size was calculated considering an alpha value of 0.05 and a difference of 1.4% in the absence of hyperemia between bromfenac and placebo.17 The protocol was approved by an ethics committee at each study center and the trial was conducted according to Good Clinical Practice and the principles of the Declaration of Helsinki. All patients provided written informed consent before the initiation of study procedures. The study was registered at ClinicalTrials.gov as NCT01591891. BODY.PATIENT POPULATION: BODY.INCLUSION CRITERIA: We enrolled 139 volunteers (bromfenac group n=69; nepafenac group n=70). Patients of both sexes (aged >18 years) with a diagnosis of cataract according to the Lens Opacities Classification System III ≤ NC4, C4 and, P4 in one eye were eligible for enrollment. Eligible patients must have had a best-corrected visual acuity of 6/60 (20/200) Snellen score. BODY.EXCLUSION CRITERIA: Patients with autoimmune diseases, history of eye surgery within 3 months prior to baseline, ophthalmological diseases, those who wear contact lenses or used any topical NSAID or corticosteroid within 2 weeks before enrollment were excluded. The presence of a condition that, in the opinion of the investigator, would have interfered with optimal participation in the study; or participation in any other clinical trial within 90 days of the screening visit were also criteria for exclusion. BODY.INTERVENTION: Patients were randomly allocated 1:1 to receive bromfenac 0.09% (Zebesten ofteno; Sophia Laboratories SA de CV, Guadalajara, Jalisco, Mexico) or nepafenac 0.1% (Nevanac; Alcon Laboratories, Inc., Fort Worth, TX, USA) using a computer that generated a list of random numbers. The morning of the scheduled cataract surgery, a nurse was instructed to instill five drops into each patient’s eye in the hour before surgery. After surgery the subjects instilled one drop of study drug topically in the conjunctival sac of the eye three times daily for 30 days. Investigators were masked to the study medication. Because the active control bottle (Nevanac) was visibly different compared to the investigational bottle, a designee at each study site, other than the investigator, was responsible for dispensing the study treatment. Attempts were made to mask the subjects by removing commercial labeling, replacing it with identical investigational labels, and packaging in identical kit boxes. BODY.ASSESSMENTS: After surgery, Visit 0 (Day 0), patients were evaluated during six study visits: Visit 1 (Day 1±1), Visit 2 (Day 7±1), Visit 3 (Day 15±1), Visit 4 (Day 30±1) and, telephone follow-up Visit 5 (Day 60±1). BODY.EFFICACY: Clinical evaluation of ocular signs: pain, photophobia, conjunctival hyperemia, chemosis, cellularity, and corneal edema were evaluated at all visits. Optical coherence tomography (OCT) was performed on Day 0, 30 and, 60 using Cirrus HD-OCT spectral domain technology, (Carl Zeiss Meditec, AG, Jena, Germany). The primary efficacy outcome was the absence of CME during spectral domain OCT determined for central retinal thickness (CRT) <275 μm, according to American Academy of Ophthalmology guidelines, after phacoemulsification. Secondary efficacy assessments included the decrement of ocular inflammation signs. BODY.SAFETY: Safety was assessed in all patients who received at least one dose of bromfenac or nepafenac during all visits. The intraocular pressure (IOP) measurement was performed using a Goldmann tonometer. The safety range was IOP >8 and <21 mmHg. Fluorescein and Lissamine green staining was considered without clinical relevance if <5% of the ocular surface was stained. Transaminase enzymes were measured at 0 and 30 days. ALT and AST were considered altered if they had an increase of two times the upper-limit from baseline. Additional safety assessments included reports of serious AEs during all visits. BODY.LABORATORY ANALYSIS: BODY.CONCENTRATION OF STUDY DRUGS: The concentration of both drugs was determined for aqueous humor sample (0.15 mL) with a 30-gauge needle on a TB syringe after completion of the paracentesis. The paracentesis was performed after first incision during the phacoemulsification. Following the aqueous humor collection, samples were stored at −40°C prior to analysis. Reversed-phase high-performance liquid chromatography (Waters Corporation, Milford, MA, USA) was performed on a Zorbax Eclipse XDB-C18 column (5 μm, 4.6×250 mm; Agilent Technologies, Santa Clara, CA, USA) using a gradient solvent system (A =0.1% formic acid in water and B =0.1% formic acid in methanol) at a flow rate of 1.0 mL/min. A photodiode array mass detector was used. Transaminase enzymes were measured in serum. The levels were determined by kinetic assays in a mass spectrophotometer. BODY.STATISTICAL ANALYSIS: The means of CRT, IOP, age and, transaminase enzymes were analyzed using a paired T-test. Ocular signs, AEs, fluorescein and lissamine dyes’ results were summarized using proportions and were analyzed with the chi-square method. The concentration of both drugs and amfenac was summarized using mean and standard deviation. In all analyses, a P-value of <0.05 (two-tailed) was considered statistically significant. All statistical analyses were conducted using SPSS software (IBM Corporation, Armonk, NY, USA) version 19. BODY.RESULTS: A total of 139 patients were enrolled in the study and completed all visits (bromfenac group n=69; nepafenac group n=70). Three sites in Mexico City included 105 patients and one site in Guadalajara, Jalisco included 34 patients. The treatment groups were comparable in regards to demographics and baseline characteristics (Table 1). The primary efficacy endpoints were similar between groups at baseline. There were no reported complications during phacoemulsification. After intervention time, the CRT did not show statistical changes – meaning CME was not present (Figure 1). There were no significant differences when comparing groups. Moreover, in both groups decreased presence of pain, chemosis, photophobia, flare, hyperemia, cellularity, and corneal edema was observed. The IOP did not increase from baseline (14.6±1.8 vs 14.2±1.8 mmHg) to final visit (14.1±1.7 vs 13.7±1.9 mmHg; P=0.650) in bromfenac and nepafenac group, respectively. The AEs were not related to the interventions. The mean peak aqueous concentration of nepafenac, amfenac, and bromfenac was 314.4±146.5, 110.2±109.0, and 207.5±152.3, respectively. There were no side effects reported after paracentesis. BODY.DISCUSSION: Inflammatory response has the purpose to repair the damaged tissues and eliminate dead cells and detritus. However this response can generate abnormalities in contiguous tissues because the cells and biomarkers involved do not have a precise limit to their action. CME presentation after phacoemulsification comes from the inflammatory response in tissues around the lens and consequently to the posterior segment structures, all this is believed to be due to the increase in the prostaglandins’ synthesis.5,18–20 Although the exact pathophysiologic mechanism of CME remains unclear, this is one of the most common causes of poor visual outcome after cataract surgery.3,4 In addition, during the recovery period, the patient can experience pain and other symptoms as a consequence of inflammatory cascade. This pathologic frame has focused the therapeutic efforts of the ophthalmologic community on prophylactic treatment with NSAIDs and corticosteroids.6,21 NSAIDs have been used for more than 3 decades, approximately, for the treatment of pain. However, due to rates of adverse effects on the ocular surface, their use has been questioned.8–10 Bromfenac is an NSAID with a particular chemical structure, a bromine atom added to the 4′ position of the aromatic ring, which allows greater penetration and concentration in ocular tissues. This pharmacokinetic profile increases its potency to inhibit COX enzymes and modulate the inflammatory response.22,23 The effectiveness of bromfenac in the treatment of CME following cataract surgery has been reported in different populations but not in Mexican subjects. The results of the present study demonstrate that bromfenac is as effective as nepafenac in preventing CME presentation. Similar to this study a trend toward increase in central retinal thickness in the nepafenac group was reported by Cable24 in a pilot study 6 weeks after surgery. However, the values were not enough to diagnose CME. The values are consistent with the findings in our study at 3 weeks. In addition, this result helps to confirm that eyes undergoing phacoemulsification experience an increase in retinal thicknesŝ5–12 μm between 4–6 weeks after surgery. The concentration levels of bromfenac in aqueous humor were similar to the reports from most authors when compared to nepafenac concentration.14,17,21,25 In the current study, the levels of nepafenac were higher than bromfenac, however despite these results, the efficacy of bromfenac was better than nepafenac as a result of its greater potency. These results confirm that from the first day of instillation of bromfenac there is a potent effect inhibiting COX-2 and reducing the pain. Similar to other authors’ findings,26–28 the signs of ocular inflammation (hyperemia, flare, cellularity, and chemosis) disappeared at the end of the intervention, even though the bromfenac concentration seemed to be low when compared to nepafenac. In spite of literature reports of adverse effects on the ocular surface epithelium due to the use of NSAIDs, our results did not show differences on lissamine green and fluorescein staining. In the bromfenac group the AEs were: CME after follow-up visits (n=1), posterior capsule rupture (n=1) during surgery, and viral conjunctivitis (n=1). In the nepafenac group, a patient presented with retinal vein occlusion and another with rhegmatogenous retinal detachment. This study adds scientific support to clinical practice about the utility of prophylactic therapy in patients undergoing phacoemulsification surgery. Further studies are necessary to measure inflammation biomarkers that could explain in more detail the pathophysiology of CME and the effect of NSAIDs on these molecules. BODY.CONCLUSION: Bromfenac 0.09% ophthalmic solution is an effective NSAID in the treatment of ocular inflammatory signs and reducing the presentation of CME after 30 days of treatment compared to nepafenac. Moreover, it is safe for ocular surface and no changes on the corneal or conjunctival epithelium were clinically detected in Mexican patients after phacoemulsification.

TITLE: No identifiable Hb1Ac or lifestyle change after a comprehensive diabetes programme including motivational interviewing: A cluster randomised trialA comprehensive diabetes programme including motivational interviewingR. Jansink et al. ABSTRACT.ABSTRACT: ABSTRACT.OBJECTIVE: To study the effectiveness of a comprehensive diabetes programme in general practice that integrates patient-centred lifestyle counselling into structured diabetes care. ABSTRACT.DESIGN AND SETTING: Cluster randomised trial in general practices. ABSTRACT.INTERVENTION: Nurse-led structured diabetes care with a protocol, record keeping, reminders, and feedback, plus training in motivational interviewing and agenda setting. ABSTRACT.SUBJECTS: Primary care nurses in 58 general practices and their 940 type 2 diabetes patients with an HbA1c concentration above 7%, and a body mass index (BMI) above 25 kg/m2. ABSTRACT.MAIN OUTCOME MEASURES: HbA1c, diet, and physical activity (medical records and patient questionnaires). ABSTRACT.RESULTS: Multilevel linear and logistic regression analyses adjusted for baseline outcomes showed that despite active nurse participation in the intervention, the comprehensive programme was no more effective than usual care after 14 months, as shown by HbA1c levels (difference between groups = 0.13; CI 20.8–0.35) and diet (fat (difference between groups = 0.19; CI 20.82–1.21); vegetables (difference between groups = 0.10; CI-0.21–0.41); fruit (difference between groups = 20.02; CI 20.26–0.22)), and physical activity (difference between groups = 21.15; CI 212.26–9.97), or any of the other measures of clinical parameters, patient's readiness to change, or quality of life. ABSTRACT.CONCLUSION: A comprehensive programme that integrated lifestyle counselling based on motivational interviewing principles integrated into structured diabetes care did not alter HbA1c or the lifestyle related to diet and physical activity. We thus question the impact of motivational interviewing in terms of its ability to improve routine diabetes care in general practice. The integration of lifestyle counselling based on motivational interviewing (MI) principles into structured diabetes care did not significantly improve diabetes care according to changes: in HbA1c or lifestyle related to diet and physical activity, or any of the other clinical parameters; in the patient's readiness to change or quality of life. Lifestyle counselling such as MI may be unsuitable for routine diabetes care in general practice. BODY.INTRODUCTION: The prevalence of diabetes is rapidly increasing, due to ageing and changes in lifestyle [1]. The situation is exacerbated by the lack of adherence to the diabetes type 2 recommendations on diet and exercise [2,3]. In many countries, such as the Netherlands, diabetes care has now largely been delegated to primary care nurses. They have to make patients aware of their unhealthy lifestyle and motivate them to change their lifestyle. The complexity of lifestyle change requires a shift from simple advice giving, as described in most diabetes guidelines, to a more patient-centred counselling-based approach [4,5]. Motivational interviewing (MI) has emerged as a promising counselling model for health promotion and disease management, even in brief encounters in general practice [6]. It is a patient-centred method, which makes the patient and professionals jointly responsible for deciding on the treatment plan [7]. Studies have shown that this method can contribute to lifestyle change, such as reducing energy intake from fat, increasing fruit and vegetable consumption [8], increasing physical activity, and lowering weight [9]. Beneficial effects on body mass index, cholesterol, and blood pressure have also been noted [6]. However, most research conducted on this topic has examined the effect of MI on a single behaviour, whereas diabetes is a complex and chronic illness that requires multiple behavioural changes. Compliance with lifestyle advice decreases when several lifestyle behaviours are targeted at the same time [10]. An effective intervention including training nurses in MI will therefore probably not be sufficient, with structured care programmes being needed [11]. Therefore, we developed a comprehensive programme with a focus on training in lifestyle counselling based on MI. In our study, we assessed the effect of this comprehensive diabetes programme on clinical parameters, lifestyle, patients’ readiness to change lifestyle and quality of life. We also evaluated the participation of nurses in the intervention programme. BODY.MATERIAL AND METHODS: BODY.STUDY DESIGN AND STUDY POPULATION: General practices were recruited to take part in a cluster randomised trial in the south-eastern part of the Netherlands, from May 2006 to February 2007. Invitation letters were sent with an estimated number of 2500 practices. The 70 practices who volunteered were visited by the first author to explain the study activities. Before randomisation 12 practices withdrew for practical reasons or anticipated disappointment not to be allocated to the intervention group. Randomisation was performed at the level of the general practice (stratified by practice size and level of urbanisation). Patients with type 2 diabetes were eligible to participate when they were younger than 80 years, had an HbA1c above 7%, and a body mass index (BMI) above 25 kg/m2. Exclusion criteria were complex comorbidity and treatment in hospital. The research team made a list of all eligible patients by screening the medical files before allocation. Patients were invited by letter and signed an informed consent form. BODY.INTERVENTION: Nurses in the intervention group received a comprehensive programme [12] consisting of (a) training in lifestyle counselling based on motivational interviewing [13]; (b) the introduction of tools for structuring diabetes care, such as training in agenda setting, a local diabetes protocol based on the national guidelines [14] that was discussed with them, and a social map for lifestyle support; (c) instruction on record keeping to integrate lifestyle counselling into general practice; and (d) introduction of tools to sustain improvements including an instruction chart (reminder) [11], regular telephone follow-ups with the target patients, a help desk that also inquired proactively about the progress of diabetes management, and a follow-up meeting for the nurses (Figure 1). Figure 1.The interventions in the comprehensive programme. Interventions (a) and (b) took place during the training sessions, which consisted of four half-day training sessions (total 16 hours) spread over the first half year. Nurses attended these sessions in groups of 5–8 outside the practice. The other activities started after these training sessions (after six months) and lasted till the post-measurement after 14 months. The record-keeping and the instruction chart were offered to nurses during the last training session. They received an oral and written explanation of the record-keeping and recommendations for regular telephone follow-ups for diabetes patients. During the intervention period the research team called the practice nurse once a quarter (three times) to enquire about their development of health counselling. The nurses could call the research team for information any time. About four months after the last training session, the nurses were invited to participate in a follow-up meeting to discuss the barriers in practice and to receive feedback about their own video-recording. What went right, and what could be better? The usual care nurses were advised to administer care consistent with current diabetes guidelines. BODY.MEASURES AND DATA COLLECTION: The primary outcomes were HbA1c and reported changes in lifestyle related to diet and physical activity. Data on diabetes outcome measures (HbA1c, blood pressure, cholesterol, BMI) and process indicators (see Table IV) were extracted from medical records. The research team identified all the eligible patients with an HbA1c of 7% or more. Only data of those patients who had given informed consent were collected by extracting the information manually from the electronic medical records. In case of more measurements per patients within the 12 months’ retrospective window, the most recent value was collected. Patient questionnaires were used to collect data on alcohol, fat, vegetables, and fruit consumption over the past month [15–17]. Physical activity was measured by asking patients to describe a typical week during the last month [18]. Physical activity was also reported more objectively based on a personal activity meter (PAM) and a diary (same week). For each lifestyle aspect the importance of and confidence in changing was rated on a five-point Likert scale; each patient's readiness to change was defined by multiplying the two items [13]. Quality of life was assessed using the Euroqol [19]. Data were gathered at baseline, and after 14 months. In the Dutch diabetes guidelines it is stated that diabetes patients should be seen once a quarter and the HbA1c should be measured yearly. For practical reasons (e.g. holidays, forgotten) it can be difficult to perform the yearly visit. We therefore took a time frame of 14 months. The exposure of nurses to MI, agenda setting, diabetes protocols, and social maps was measured by recording their attendance at the different training sessions on these subjects. Furthermore, we asked nurses if they used the instruction chart, and we recorded the number of nurses who received three telephone follow-ups from the research team, as well as their participation in the follow-up meeting. BODY.SAMPLE SIZE: With the intervention used, we expected 50% of the eligible patients to achieve an Hba1c below 7% [12]. Based on an alpha of 5% and a beta of 80% a random sample of 30 general practices with five patients each was needed, taking into account an intra-cluster correlation of 0.05 (patients in practices). For lifestyle we expected a 5% change in usual care, and an extra 10% change due to the intervention [12]. Without changing the other assumptions, a total of 68 practices with 10 patients were needed to detect the expected difference in lifestyle between the intervention and usual care groups. We thus planned to recruit 70 general practices with 700 patients, allowing for some loss. BODY.STATISTICAL ANALYSIS: Means and standard deviations, and percentages where appropriate, were used to summarise the characteristics of the general practices, nurses, and patients. Comparisons between the intervention and usual care arms were adjusted for clustering within practices. Continuous outcome measures (clinical outcomes, reported aspects of lifestyle, and quality of life) were analysed with multilevel linear regression in SPSS. The HbA1c was added as a continuous measure in the statistical model to avoid loss of power. Binary outcome measures (patients’ readiness to change and diabetes process indicators) were analysed using multilevel logistic regression in SAS. In these analyses baseline measures were defined as a separate predictor in the model. We also adjusted the models for baseline characteristics (from practices, nurses, or patients) that differed significantly between the intervention and usual care groups. BODY.RESULTS: BODY.STUDY POPULATION: Figure 2 presents the number of general practices and participants in this trial. Table I presents the baseline characteristics of the general practices, nurses, patients, and the baseline values regarding outcome measures, lifestyle, and quality of life. Figure 2.Flow diagram of general practices and patients at different stages (enrolment, allocation, baseline measurement, follow-up, and analysis) of the trial. Table I.Baseline characteristics of general practices, primary care nurses, patients, and baseline values of measures. Intervention Usual care SD SD General practices (n = 53):a 25 28 Mean number of patients in general practiceb 4566 2703.8 5657 4153.3 Mean number of patients with type 2 diabetes in general practice 195 109.6 202 119.4 Mean FTEc primary care nurses in general practice 0.6 0.3 0.8 0.6 Primary care nurses (n = 53):d 25 28 Mean age in years 40.7 7.8 44.4 6.6 Number of men/number of woman 2/23 1/27 Mean years of experience with diabetes consultations 3.6 2.1 3.6 2.1 Nurses who were formerly practice assistants 48.0 53.6 Mean years of experience as practice assistant 4.3 5.3 8.4 8.8 Patients (n = 521): 229 292 % men 55.9 54.0 Mean age in years 64.1 8.9 63.9 9.8 Mean duration of diabetes in years 7.5 6.0 7.8 5.8 Baseline (n = 336): 134 202 Outcome measures: HbA1c, % 7.8 0.9 7.7 0.7 Systolic blood pressure, mm Hg 144.4 20.3 140.7 18.0 Diastolic blood pressure, mm Hg 81.9 10.6 79.9 9.9 LDL, mmol/l 2.8 1.0 2.5 0.8 Total cholesterol, mmol/l 4.7 1.0 4.5 1.0 BMI, kg/m2 30.7 4.2 30.7 4.2 Lifestyle: Alcohol, units/daye 2.3 1.0 2.2 1.1 Fat score, g/day 14.1 4.5 14.5 4.9 Vegetables, tablespoons/day 2.9 1.6 3.0 1.5 Fruit, pieces/day 1.9 1.1 1.8 1.1 Physical activity, minutes/day 64.8 66.1 58.6 45.1 Pam scoref 19.2 8.5 21.2 8.0 Low activity, minutes/day 73.2 40.3 77.0 37.0 Medium activity, minutes/day 21.1 23.4 23.4 23.8 High activity, minutes/day 0.3 0.8 0.7 3.3 Diary activity, minutes/day 129.9 77.4 153.9 103.4 Quality of life: VAS score 74.1 16.2 73.1 13.5 Notes: aFour general practices withdrew from the study before the nurse had completed the questionnaire about the baseline characteristics. bSD = standard deviation. cFTE = full time equivalent; dIn six practices two nurses were employed. In such cases we calculated the mean of the nurse characteristics, because the mean values were used in follow-up analyses. eOnly people who reported alcohol consumption. fPam score = Personal activity meter score. BODY.FOLLOW-UP ANALYSES: Based on the medical records of the dropouts at baseline we learned that follow-up was somewhat higher among older patients (63.8 vs. 61.4 years), and patients with a lower BMI (30.8 vs. 32.4) and a lower HbA1c (7.8% vs. 8.0%). At follow-up after 14 months we lost very little information from the medical records, but again not all patient questionnaires were returned (see Figure 2). The follow-up was not affected by age or BMI, but the HbA1c of the non-responders was slightly higher (0.2%) than that of responders. BODY.DIABETES CARE: Table II shows that compared with usual care the comprehensive programme did not result in statistically significant improvements in the diabetes outcome measures (HbA1c, blood pressure, cholesterol, BMI). However, the small changes in cholesterol outcomes (LDL and total) had a p < 0.10. Post measurement showed that in the intervention group 34.5% reached the HbA1c target value (below 7%) compared with 34.0% in the usual care group (OR = 1.17, 95% CI = 0.74–1.85, p-value = 0.49). The number of people with an HbA1c above 8.5% decreased in the intervention group from 16.5% to 9.8% and in the usual care group from 11.9% to 9.8% (OR = 1.01, 95% CI = 0.42–2.39, p-value = 0.99). The comprehensive diabetes programme was no more effective than usual care in terms of the reported consumption of alcohol, fat, vegetables and fruit, or physical activity. Physical activity showed different outcomes when measured by questionnaire, personal activity meter, or diary, but none of these outcomes differed between intervention and usual care. The intervention did not increase or decrease quality of life compared to usual care. Table II.Effect of comprehensive diabetes programme on diabetes outcome measures, lifestyle, and quality of life after 14 months’ follow-up.a Intervention Usual care Difference between groupsb 95% CI p-value M SD n M SD n Outcome measures: HbA1c, % 7.3 0.7 129 7.4 1.0 197 0.13 −0.08 – 0.35 0.221 Systolic blood pressure, mm Hg 141.5 17.0 120 137.8 15.8 185 −1.98 −5.63 – 1.67 0.279 Diastolic blood pressure, mm Hg 79.5 8.4 120 77.6 9.2 120 −1.17 −3.41 – 1.07 0.294 LDL, mmol/l 2.6 0.8 106 2.4 0.6 178 −0.15 −0,32 – −0.02 0.081 Total cholesterol, mmol/l 4.5 1.0 122 4.2 0.8 186 −0.21 −0.41 – 0.00 0.051 BMI, kg/m2 30.2 4.0 106 30.5 4.6 179 0.36 −0.19 – 0.90 0.198 Lifestyle: Alcohol, units/dayc 2.2 1.0 58 2.2 1.1 95 0.04 −0.14 – 0.23 0.647 Fat score, g/day 13.9 5.4 105 14.2 6.1 163 0.19 −0.82 – 1.21 0.708 Vegetables, tablespoons/day 3.1 1.6 102 3.1 1.5 165 0.10 −0.21 – 0.41 0.518 Fruit, pieces/day 1.8 1.1 119 1.7 1.2 173 −0.02 −0.26 – 0.22 0.884 Physical activity, minutes/day 62.8 69.6 124 59.1 51.3 171 −1.15 −12.26 – 9.97 0.839 Pam scored Low activity, minutes/day 78.3 40.3 78 78.8 39.8 120 −2.70 −10.52 – 5.14 0.498 Medium activity, minutes/day 22.5 27.0 78 22.6 20.2 120 −1.46 −6.80 – 3.89 0.592 High activity, minutes/day 0.4 0.9 78 1.0 6.5 120 0.18 −0.65 – 1.01 0.669 Diary activity, minutes/day 152.9 97.6 84 157.4 89.0 128 −19.36 −39.97 – 1.26 0.066 Quality of life: VAS score 75.3 16.2 111 73.5 13.6 171 −1.27 −4.50 1.97 0.441 Notes: aAdjusted for baseline measures, nurses’ years of experience, and cluster effects. bDifference between intervention and usual care group (reference group). cOnly people who reported alcohol consumption. dPam score = Personal activity meter score. Table III indicates that the intervention did not change any aspect of patients’ readiness to change their lifestyle. The number of participants who themselves reported not meeting the norm was lower than expected in a group with a BMI above 25 kg/m2. Table III.Effect of comprehensive diabetes programme on patient's readiness to change lifestylea after 14 months’ follow-up.b Intervention Usual care M SD n M SD n B 95% CI p-value Alcohol, units/weekc 12.8 4.1 11 10.5 3.6 18 −1.03 −3.93 – 1.86 0.471 Fat, g/day 14.8 6.0 30 13.0 3.9 43 0.21 −3.19 – 3.60 0.901 Vegetables, g/day 12.5 5.1 89 13.7 4.5 150 0.74 −0.47 – 1.96 0.228 Fruit, pieces/day 12.5 4.4 71 11.9 4.1 116 −0.38 −1.82 – 1.05 0.597 Physical activity, minutes/day 11.7 5.4 63 11.1 4.1 126 −0.48 −2.13 – 1.17 0.563 Notes: aOnly for patients who did not reach the norm for lifestyle; range 1–25. bAdjusted for baseline measures and nurses’ years of experience. cOnly people who reported alcohol consumption. Table IV.Effect of comprehensive diabetes programme on diabetes process indicators after 14 months’ follow-up.a Number of patients (%) Interventionn = 186 Usual caren = 263 Odds ratio 95% CI p-value Dietary advice 104 (56) 119 (45) 0.96 0.86 – 1.06 0.838 Physical activity advice 106 (57) 116 (44) 0.96 0.87 – 1.06 0.984 HbA1c checked 182 (99) 251 (95) 2.13 0.60 – 7.53 0.239 Blood pressure checked 185 (100) 245 (93) 13.59 1.79 – 103.37 0.01 Total cholesterol checked 174 (94) 242 (92) 0.99 0.94 – 1.04 0.554 LDL cholesterol checked 169 (91) 239 (91) 2.25 1.01 – 5.00 0.838 Creatinine (serum) checked 174 (94) 242 (92) 1.02 0.96 – 1.07 0.322 Microalbuminuria checked 163 (88) 223 (85) 0.98 0.93 – 1.04 0.931 Eye examinationb 61 (33) 106 (40) 1.01 0.96 – 1.06 0.717 Foot examination 131 (70) 205 (78) 0.98 0.94 – 1.02 0.101 BMI determined 166 (89) 230 (87) 0.97 0.91 – 1.04 0.832 Cholesterol-lowering medication 80 (43) 93 (36) 0.97 0.88 – 1.07 0.955 Notes: aAdjusted for baseline measures and nurses’ years of experience. bIn general once in 24 months, but eye examinations were recorded in the last 12 months. The findings on diabetes process indicators revealed that the number of patients receiving lifestyle advice neither increased nor decreased as a result of the comprehensive diabetes programme (Table IV). The probability of annual checks on HbA1c, blood pressure, and LDL increased, but only significantly for blood pressure. However, the high adherence rates at baseline in both the intervention and usual care group suggested that this significant finding is not clinically relevant. Nurses in the intervention actively participated in the programme, with 93% attending at least three out of four MI training sessions. The social maps and local diabetes protocols were discussed by 74% of the nurses. All practices received the three quarterly telephone follow-ups from the research team after training. During these conversations, nurses indicated that the instruction chart was very useful. Most of them had the chart on their desk and used it during or after consultations. Although the nurses requested an MI follow-up meeting, participation was low (37%). BODY.DISCUSSION: BODY.STATEMENT OF PRINCIPAL FINDINGS: The comprehensive diabetes programme had no effect on HbA1c or reported aspects of lifestyle, nor on the other diabetes outcome measures or quality of life. Patients with type 2 diabetes were no more ready to change lifestyle in the intervention practices than in usual care, and the adherence of nurses to guidelines for process measures showed no relevant improvement, despite their active participation in the training programme. BODY.STRENGTHS AND LIMITATIONS OF THE STUDY: Strengths include the cluster RCT design, and the variety of measures (outcome and process, subjective and objective). As 70 out of 2500 practices participated voluntarily, it can be assumed that only practices that were enthusiastic about improving diabetes care by using MI were recruited. This could have affected the study results. However, improvement was not shown in the intervention or in the control group. A limitation of the study is the loss to follow-up in the lifestyle measures from the patient questionnaire. At baseline and follow-up we lost participants with a relatively higher HbA1c than those who remained within the study, which narrowed the room for improvement in HbA1c. We also noticed that the proportion of measurements differed between the intervention and usual care group. Although we formulated strict inclusion criteria and performed randomisation, it is possible that the population in the intervention and usual care group differed on variables beyond our set of measurements. Another limitation was the underpowered nature of the patient questionnaires. However, based on the difference between groups, confidence intervals, and p-values in Table II, we can assume that a larger sample size would not have led to any significant changes either. The follow-up period was 14 months. It is theoretically possible that the effect can only be seen in the long term. But this contradicts other studies that showed an effect of MI directly after its introduction [6,20]. BODY.COMPARISON WITH EXISTING LITERATURE: The comprehensive diabetes programme was based on elements that had proved to be effective such as structured diabetes management [11] and motivational interviewing [6,8,9]. Previous research has reported that for patients with type 2 diabetes the use of MI can improve glucose control, dietary changes, smoking, weight, physical activity, motivation for lifestyle change, and adherence to diabetes guidelines [6,8,9,21]. Knowing that dietary advice and physical activity are associated with a lower HbA1c [22,23], we would have expected our comprehensive diabetes programme to have an effect on HbA1c and lifestyle. However, recently more studies have questioned the effectiveness of MI in terms of clinical outcomes, lifestyle, quality of life, and self-efficacy for patients with type 1 and 2 diabetes or in cardiovascular risk management in routine care in general practice [24–27]. BODY.EXPLANATION OF THE FINDINGS: There are several possible reasons why our trial failed to demonstrate any effectiveness of the comprehensive diabetes programme. A thought could be that Dutch diabetes care is already on a high level as the guideline was introduced in 1989, which makes further improvement difficult as could be shown by the mean HbA1c of 7.8% at baseline. Improvement on BMI, however, was certainly possible in the study population. Another explanation could be that the education of the nurses was of such level that lifestyle education was performed on almost the same level in both the intervention and control group, and the training programme hardly added value. Nurses in the Netherlands are trained in a three- to four-year curriculum (middle or higher education) and afterwards they can specialise in primary care following a one- or two-year curriculum. Lifestyle counselling is part of the curriculum, but not specifically focused on motivational interviewing. The education is a prerequisite for effective lifestyle counselling, but no guarantee for reaching good outcomes. The lifestyle outcomes in the study showed that there could be improvement. We could consider the quality of our training programme itself, but there are no comparable data. It is only known that the four training sessions offered were sufficient [28]. As nurses themselves asked for an extra session, it can be concluded that more support was desirable. Perhaps training on the job can help to produce more effective lifestyle counselling [29]. As room for improvement was available and nurses in the intervention group were better equipped to perform lifestyle counselling, the study results could be explained by the study design itself. Some of its weaknesses have already been discussed, but there impact seems to be limited. Could it be that the time frame chosen was too short? Fourteen months after the intervention the post measurements took place, but not all of the patients were seen at the same time. An extra analysis on this subject showed that on average the measurements were performed in the midst of the time frame. It can be assumed that lifestyle changes take more time, but earlier studies have found MI effects after 3–4 months [6,30]. Explanations can also be found in our target population. MI was originally developed for substance abuse [13], requiring a single behavioural change, whereas diabetes is a complex chronic illness that requires multiple behavioural changes. MI may be less effective for multiple behavioural changes, despite the fact that our nurses were trained to set the agenda. In case of diabetes it may be better to organise a setting that is explicitly dedicated to MI. Separate MI sessions have been shown to be successful [31]. In our programme the counselling strategy was applied during regular nurse consultations. Or are we being naive? Targeting changes in biomedical parameters and lifestyle in an elderly population with type 2 diabetes is a battle involving a complicated social, psychological, and physiological web of related issues [32]. Our comprehensive diabetes programme may not have been nearly comprehensive enough. BODY.IMPLICATIONS FOR FUTURE RESEARCH ON CLINICAL PRACTICE: Our results indicate a need for further research on lifestyle counselling embedded in primary care and the assessment of factors influencing the use of such counselling strategies for a better understanding of the applicability of interventions in diabetes care. One approach may lie in the argument that the environment in which we live may be the driving force behind many of our less healthful lifestyle habits [33]. A health protection approach with a possible role for the polypill to reduce cardiovascular risk may be more effective than a motivational intervention [34–36]. Another direction can be an investment in acquiring knowledge on personalised lifestyle counselling. Is it possible to customise lifestyle counselling based on genetic or other information? In this case MI will be offered to those patients who will benefit most instead of being part of routine diabetes care. Nurses can focus energy into work for this group with potentially more fruitful results. BODY.FUNDING: This study was funded by ZonMW – the Netherlands Organization for Health Research and Development, 945-16-113. BODY.ETHICS APPROVAL: The medical ethics committee of the University Medical Centre Nijmegen approved the study. Current Controlled Trials ISRCTN68707773.

TITLE: Comparing biosimilar SB2 with reference infliximab after 54 weeks of a double-blind trial: clinical, structural and safety results ABSTRACT.ABSTRACT: ABSTRACT.OBJECTIVES: SB2 is a biosimilar to the reference infliximab (INF). Similar efficacy, safety and immunogenicity between SB2 and INF up to 30 weeks were previously reported. This report investigates such clinical similarity up to 54 weeks, including structural joint damage. ABSTRACT.METHODS: In this phase III, double-blind, parallel-group, multicentre study, patients with moderate to severe RA despite MTX were randomized (1:1) to receive 3 mg/kg of either SB2 or INF at 0, 2, 6 and every 8 weeks thereafter. Dose escalation by 1.5 mg/kg up to a maximum dose of 7.5 mg/kg was allowed after week 30. Efficacy, safety and immunogenicity were measured at each visit up to week 54. Radiographic damage evaluated by modified total Sharp score was measured at baseline and week 54. ABSTRACT.RESULTS: A total of 584 patients were randomized to receive SB2 (n = 291) or INF (n = 293). The rate of radiographic progression was comparable between SB2 and INF (mean modified total Sharp score difference: SB2, 0.38; INF, 0.37) at 1 year. ACR responses, 28-joint DAS, Clinical Disease Activity Index and Simplified Disease Activity Index were comparable between SB2 and INF up to week 54. The incidence of treatment-emergent adverse events and anti-drug antibodies were comparable between treatment groups. Such comparable trends of efficacy, safety and immunogenicity were consistent from baseline up to 54 weeks. The pattern of dose increment was also comparable between SB2 and INF. ABSTRACT.CONCLUSION: SB2 maintained similar efficacy, safety and immunogenicity with INF up to 54 weeks in patients with moderate to severe RA. Radiographic progression was comparable at 1 year. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov (http://clinicaltrials.gov; NCT01936181) and EudraCT (https://www.clinicaltrialsregister.eu; 2012-005733-37) Rheumatology key messages SB2 is a monoclonal antibody biosimilar to the reference infliximab. SB2 demonstrated similar efficacy, safety and immunogenicity compared with reference infliximab up to 54 weeks. SB2 showed comparable rates of radiographic progression up to 54 weeks compared with reference infliximab in RA. BODY.INTRODUCTION: Biological DMARDs (bDMARDs), including TNF-α inhibitors, have changed the paradigm of the treatment of rheumatic diseases such as RA, AS and PsA [1–3]. bDMARDs have shown significant efficacy in patients who do not respond to conventional synthetic DMARDs alone [4, 5], however, the high cost of these agents is often considered a barrier for widespread use. The introduction of biosimilar DMARDs (bsDMARDs), which are less costly than the reference products, may help to contain health care costs and there is great anticipation that bsDMARDs will make bDMARDs substantially more accessible to patients who are in need of such treatment but currently cannot access them for cost reasons [6–9]. A biosimilar is a biological medicinal agent that contains a similar active substance as an approved biological medicinal product, also referred to as the reference or originator product, and is intended to be used in the same manner as the reference or originator product [10]. As an exact copy of the reference product is not feasible, a biosimilar must be similar in terms of quality characteristics, biological activity, pharmacokinetics, safety, immunogenicity and efficacy [11]. The rigorous process involved in proving the biosimilarity of a proposed biosimilar to its reference product is detailed in major regulatory guidelines in the European Union (EU) and the USA [12, 13]. SB2 is a biosimilar to the infliximab (INF) reference product Remicade (Janssen Biotech, Horsham, PA, USA), a chimeric human-murine mAb that is specific to human TNF-α and approved for the treatment of various rheumatic diseases such as RA, AS and PsA as well as non-rheumatic diseases such as psoriasis, Crohn’s disease and ulcerative colitis [14]. SB2 has been evaluated through various biosimilar comparability studies, including quality, pharmacokinetic and phase III clinical studies, to prove biosimilarity and is now approved under the names Flixabi and Renflexis [15, 16]. We have previously reported the primary results of a 30 week phase III clinical study conducted in patients with RA that showed equivalent efficacy (measured by the ACR 20% response rate), comparable safety, immunogenicity and similar pharmacokinetic profiles between SB2 and INF [16]. While these findings significantly support the biosimilarity of SB2 to INF, long-term clinical trial data are needed to further demonstrate biosimilarity in terms of efficacy and safety [13]. Part of the efficacy analysis is the provision of evidence on biosimilarity regarding inhibition of joint damage, an important attribute of TNF blockade that was not part of the 30 week study of SB2 [17, 18]. Thus the objective of this study was to compare the radiographic progression of structural joint damage up to 54 weeks between SB2 and INF, as well as to investigate whether the comparable clinical efficacy, safety and immunogenicity of SB2 to INF as observed up to 30 weeks was maintained and remained comparable up to 54 weeks. BODY.METHODS: BODY.PATIENTS AND STUDY DESIGN: The details of this phase III study have been reported previously [16]. In brief, this study was a phase III, randomized, double-blind, parallel-group, multicentre clinical study conducted at 73 investigator sites in 11 countries in Europe and Asia (NCT01936181; EudraCT 2012-005733-37). Patients who were 18–75 years of age, biologic naïve and diagnosed with moderate to severe RA, defined as six or more swollen and tender joints, ESR ⩾28 mm/h or serum CRP ⩾1.0 mg/dl at screening, despite MTX therapy for at least 6 months were enrolled in the study. Patients were randomized according to a computer-generated and interactive web responsive system (Cenduit, Bangalore, India) in a 1:1 ratio to receive either SB2 3 mg/kg or EU-sourced INF 3 mg/kg via i.v. infusion. Patients received either SB2 or INF during each visit at weeks 0, 2, 6, 14, 22, 30, 38 and 46. Starting at week 30, stepwise dose increments by 1.5 mg/kg up to a maximum of 7.5 mg/kg were permitted at each visit if the patient’s RA symptoms were not well controlled by the existing dose. This dose modification scheme is in accordance with the EU Remicade label (i.e. summary of product characteristics) [19]. The assessment of RA symptom control was dependent on the investigator’s clinical judgement. All patients included in the study were required to receive a stable dose of oral or parenteral MTX (10–25 mg/week) and folic acid (5–10 mg/week) and had moderate to severe active disease despite MTX therapy. NSAIDs and glucocorticoids (equivalent to ⩽10 mg prednisolone) were permitted if the patient was on a stable dose for at least 4 weeks prior to randomisation. Pre-medications for infusion-related reactions, such as paracetamol, antihistamines or corticosteroids, were allowed per the investigator’s discretion. All patients were evaluated for tuberculosis (TB) through medical history, chest X-ray, and QuantiFERON-TB Gold tests at screening and weeks 22 and 54. This study was conducted in accordance with the International Conference on Harmonisation Good Clinical Practice guidelines and the Declaration of Helsinki. All patients provided formal written informed consent prior to participating in the study. BODY.ASSESSMENTS: Efficacy, safety and immunogenicity assessments were conducted at each study visit for all patients prior to SB2 or INF infusion. The clinical efficacy endpoints included 20, 50 and 70% ACR (ACR20, ACR50, ACR70) responses and 28-joint DAS (DAS28) scores. The ACR response also includes a patient-reported outcome of physical function, the HAQ Disability Index [20]. In addition, the Simplified Disease Activity Index (SDAI) and Clinical Disease Activity Index (CDAI) were employed to assess low disease activity (LDA) or remission status [21, 22]. Structural joint damage was assessed by the van der Heijde modified total Sharp score (mTSS), composed of erosion and joint space narrowing scores [23]. The radiographic images were evaluated by two independent readers who were blinded to patient identity, treatment and the time of measurement. Progression of joint damage was calculated as the mean difference between the baseline and the week 54 measurements (i.e. the mean change in the mTSS). When the change score was within the top 5% of cases with the highest differences in score between readers, the radiographs required consensus review by the primary readers. Safety endpoints included treatment-emergent adverse events (TEAEs), serious AEs and AEs of special interest (defined as serious infections or active TB). Abnormalities in clinical laboratory values and vital signs were also assessed. Immunogenicity endpoints such as the incidence of anti-drug antibodies (ADAs) and neutralizing antibodies (NAbs) were measured. Patients with at least one ADA-positive result following the randomization visit were identified as ADA positive [24]. Those patients who were ADA positive were also assessed for NAbs. To assess immunogenicity, a single-assay approach with an SB2 tag was used. Validated electrochemiluminescence immunoassays were used to measure ADAs and NAbs using a competitive ligand-binding assay (Meso Scale Discovery platform, Meso Scale Discovery, Rockville, MD, USA). Pharmacokinetic parameters were assessed up to week 30 and have been previously reported [16]. BODY.SAMPLE SIZE AND STATISTICAL ANALYSIS: Sample size calculation, as described previously, was based on comparing the primary endpoint defined as the ACR20 response rate at week 30 in the per-protocol set (PPS), assuming an equivalence margin of ±15% and a dropout rate of 20% [16]. Based on these assumptions, 584 patients were required. Besides the PPS, a full analysis set (FAS), which follows the principles of intention-to-treat analysis, was also used in efficacy outcome analyses. Safety outcomes were analysed in the safety set (SAF), which consisted of patients who received at least one dose of either SB2 or INF. As the primary endpoint of the study was met, this report aimed to compare long-term efficacy (including radiographic progression), safety and immunogenicity between SB2 and INF up to week 54. Analyses of ACR responses were conducted using both the PPS and FAS, while DAS28, CDAI and SDAI analyses were conducted using only the FAS. All FAS analyses were performed on patients who had data on that time point (i.e. as observed analysis). Treatment differences in ACR response rates between SB2 and INF were estimated in a similar manner as in the 30 week report, adjusted by CRP and geographical region, with 95% CIs. Safety analyses were conducted by comparing the frequency of TEAEs, laboratory abnormalities and serious AEs reported up to week 54 from the SAF. Immunogenicity was also analysed from the SAF. When applicable, subgroup analysis of efficacy or safety outcomes was performed by ADA status (ADA positive or ADA negative up to week 54). Statistical analysis was performed using SAS version 9.2 (SAS, Cary, NC, USA). When statistical testing was required, statistical significance was determined as P < 0.05. BODY.RESULTS: BODY.PATIENTS: As previously reported, from 805 patients screened, 584 patients were randomized to receive study treatment. Of these, 583 patients received at least one infusion of SB2 or INF and were included in the FAS and SAF. The patient disposition was similar between the SB2 and INF treatment groups; 78.0% of the SB2 treatment group and 76.8% of the INF treatment group completed the 54 week study (Fig. 1). Baseline characteristics have been previously reported as comparable between the two treatment groups and are provided in Supplementary Table S1, available at Rheumatology Online. Among the baseline characteristics, efficacy components such as tender or swollen joint count, visual analogue scale and HAQ scores and the progression at weeks 30 and 54 are also reported, which show comparable improvement between the two treatment groups. Fig. 1Disposition flow chart of the study population Eight patients’ data from sites in Eastern Ukraine were excluded from the analysis due to regional issues (n = 4 in SB2, n = 4 in INF). INF: reference infliximab. BODY.EFFICACY: Radiographic progression from baseline to week 54 is shown in Fig. 2. The mean change from baseline in mTSS at week 54 was numerically comparable between treatment groups (SB2, 0.38; INF, 0.37). At week 54, the adjusted mean difference of change from baseline in mTSS was 0.01 (95% CI −0.53, 0.56), suggesting a similar rate of radiographic progression between SB2 and INF. Also, the distribution of the cumulative probability plots was similar. When analysing the components of mTSS, the mean change from baseline in erosion score was 0.14 for SB2 and −0.03 for INF and the mean change from baseline in joint space narrowing score was 0.24 and 0.40, respectively (Supplementary Table S2, available at Rheumatology Online). Fig. 2Cumulative probability of change in the mTSS at week 54 (full analysis set) INF: reference infliximab. Disease activity measured by DAS28, CDAI and SDAI and classification by LDA or remission are shown in Fig. 3. The pattern of improvement over time was highly similar on all disease activity indices up to 54 weeks (mean DAS28 at week 54, 4.05 in both SB2 and INF). When disease activity was categorized into LDA and remission, the proportion of patients who achieved either LDA or remission was similar between SB2 and INF at week 54 (45.8% of SB2- and 47.1% of INF-treated patients achieved LDA or remission by the CDAI and 46.9% of SB2- and 49.5% of INF-treated patients achieved LDA and remission by the SDAI). Fig. 3Improvement of disease activity and remission rates (full analysis set) (A) Mean DAS28, CDAI and SDAI up to week 54. (B) Disease activity classification (remission and LDA). Remission is defined as DAS28 <2.6, CDAI ≤2.8 or SDAI ≤3.3 and LDA is defined as DAS28 ≥2.6–<3.2, CDAI ≤10.0 or SDAI ≤11.0. The data above each bar are the total sum of remission and LDA. INF: reference infliximab. ACR response rates were similar between treatment groups up to week 54 for both PPS and FAS (Fig. 4). The ACR20 response rate at week 54 in the PPS was 65.3% for SB2 and 69.2% for INF, with an estimated treatment difference of −3.07% (95% CI −12.00, 5.86). This similarity was demonstrated once more in the FAS; the ACR20 was 64.5% for SB2 and 68.4% for INF, with an estimated treatment difference of −3.34% (95% CI −11.86, 5.18). The overall efficacy did not differ from what had been observed at week 30 (ACR20 responses in PPS: 64.1% in SB2 and 66.0% in INF; ACR20 responses in FAS: 63.6% in SB2 and 65.3% in INF). Fig. 4ACR20, 50 and 70 response rates up to week 54 (full analysis set) INF: reference infliximab. SB2 or INF dose increases occurred from week 30 per investigators’ judgement of the patient’s RA disease activity. The pattern of dose increases is shown in Supplementary Table S3, available at Rheumatology Online. Approximately 35% of the study population had undergone at least 1 cycle of a dose increase. The pattern of dose increments was comparable between the SB2 and INF treatment groups. The mean dose at the last infusion visit (week 46) was 3.74 mg/kg for SB2 and 3.72 mg/kg for INF. The relationship between dose increment and efficacy are shown in Supplementary Fig. S1, available at Rheumatology Online. Those who had at least one dose increase had baseline (week 30) lower ACR20 response rates compared with those who never had a dose increase. A higher ACR20 response compared with baseline was observed in patients who had at least one dose increase at week 54 compared with week 30 and the ACR20 response rates were comparable between SB2 and INF. BODY.SAFETY: SB2 was well tolerated during the study. The incidence of total and commonly occurring TEAEs, serious AEs and TEAEs of special interest were comparable between the SB2 and INF treatment groups up to week 54 (Table 1). Most TEAEs were reported as mild to moderate in severity. The most commonly reported TEAEs were latent TB, nasopharyngitis and an increase in alanine aminotransferase. The majority of patients with latent TB received TB prophylaxis, and none of these patients developed active TB during the study. The incidence of active TB was the same as in the 30 week report (one case in both SB2 and INF); no new cases occurred thereafter up to week 54. This was also the case with malignancies, congestive heart failure and death. One new case of serious infection (diabetic foot infection) developed in the INF treatment group. The incidence of infusion-related reactions (IRRs) was comparable between the two treatment groups [SB2, n = 17 (5.9%); INF, n = 15 (5.1%)] and of those cases, five were considered serious (SB2, n = 3; INF, n = 2). In summary, the safety profile of SB2 remained relatively consistent with previously reported data (up to 30 weeks) and was comparable to that of INF. Table 1Summary of TEAEs up to week 54 Type of TEAESB2 (n = 290)INF (n = 293)Any TEAEs, n (%) 179 (61.7) 191 (65.2) Common TEAEs of incidence ≥2%, n (%)     Latent tuberculosis 19 (6.6) 21 (7.2)     Nasopharyngitis 18 (6.2) 20 (6.8)     Alanine aminotransferase increased 23 (7.9) 9 (3.1)     RA 20 (6.9) 11 (3.8)     Headache 16 (5.5) 13 (4.4)     Upper respiratory tract infection 12 (4.1) 11 (3.8)     Aspartate aminotransferase increased 12 (4.1) 10 (3.4)     Bronchitis 9 (3.1) 13 (4.4)     Back pain 7 (2.4) 11 (3.8)     Arthralgia 8 (2.8) 8 (2.7)     Pneumonia 7 (2.4) 8 (2.7)     Urinary tract infection 8 (2.8) 6 (2.0)     Hypertension 5 (1.7) 9 (3.1)     Cough 6 (2.1) 7 (2.4)     Rash 6 (2.1) 6 (2.0)     Pharyngitis 5 (1.7) 7 (2.4)     Pyrexia 3 (1.0) 8 (2.7)     Abdominal pain upper 4 (1.4) 6 (2.0)     Dizziness 2 (0.7) 6 (2.0)     Dyspepsia 1 (0.3) 7 (2.4) Any serious TEAEs 29 (10.0) 31 (10.6) Serious infections or tuberculosis 9 (3.1) 7 (2.7) Infusion-related reactionsa 17 (5.9) 15 (5.1) Malignancyb 2 (0.7) 0 (0.0) Deathc 0 (0.0) 1 (0.3) aFive cases were serious (two cases of hypersensitivity and one case of anaphylactic reaction in SB2 and one case of anaphylactic shock and one case of urticaria in INF). bBreast cancer and prostate cancer. cRelated to congestive heart failure. BODY.IMMUNOGENICITY: Immunogenicity was comparable between SB2 and INF with no statistically significant difference. The proportion of ADA-positive patients up to week 54 was 62.4% for SB2 and 57.5% for INF (P = 0.270), a trend consistent with the previous comparable 30 week report (SB2, 55.1%; INF, 49.7%; P = 0.212). The proportion of patients with NAbs among the patients who developed ADA was also comparable between the two treatment groups (92.7% for SB2 and 87.5% for INF). An analysis of efficacy and safety by ADA status is shown in Fig. 5. ACR20 response rates at week 54 were comparable between SB2 and INF within each ADA subgroup, with higher responses in ADA-negative patients than in ADA-positive patients (Fig. 5A). The ACR20 response rate at each visit by ADA subgroup is shown in Supplementary Fig. S2, available at Rheumatology Online. ACR20 responses were generally comparable between the SB2 and INF treatment groups among patients who had overall negative or positive ADA results up to week 54. Fig. 5Analysis of ACR20 response rate and infusion-related reaction incidence by 54 week ADA status (A) ACR20 response rate at week 54 in the PPS set by 54 week overall ADA status. (B) The patients with infusion-related reaction up to week 54 in the SAF set by 54 week overall ADA status. INF: reference infliximab. Since IRRs are known to be associated with positive ADA status, the incidence of IRRs was analysed by ADA status. As expected, the incidence of IRRs was higher in patients who were ADA positive than in those who were ADA negative and the incidence was comparable between the treatment groups within each ADA subgroup up to 54 weeks [15 (8.4%) for SB2, 11 (6.5%) for INF in ADA-positive patients; 2 (1.9%) for SB2, 4 (3.2%) for INF in ADA-negative patients; Fig. 5B]. BODY.DISCUSSION: The results of this study demonstrate that the similarity in efficacy, safety and immunogenicity previously reported in the SB2 and INF treatment groups was maintained up to 54 weeks in patients with RA. In particular, structural joint damage measured by radiographic progression was comparable between SB2 and INF at 1 year. The degree of radiographic progression was also comparable to the pivotal ATTRACT study [17]. In addition, this report provides data related to increasing infliximab doses and has also demonstrated comparable efficacy profiles between SB2 and INF. All of these findings strongly support the biosimilarity of SB2 to INF over the long term. Since biologics are used in the treatment of rheumatic diseases and can be chronically used [25], long-term clinical trial data that prove biosimilarity to reference products may further increase the confidence in prescribing biosimilars. Radiographic progression has been measured in major pivotal trials of biologics for the treatment of RA as an index of long-term efficacy [17, 26]. In this study, the progression of joint disease, determined by the mTSS, suggests that the rate of radiographic progression is comparable between the SB2 and INF treatment groups. TNF inhibitors are thought to decouple the association between inflammation and joint damage [27-29], and thus even if disease activity is inadequately controlled with anti-TNF therapy, radiographic progression is still inhibited. The similarity in inhibition of joint damage progression observed with SB2 compared with INF further augments evidence of the biosimilarity between SB2 to INF on a long-term structural basis, in addition to disease activity. In this study, SB2 and INF maintained comparability up to 54 weeks in all efficacy outcomes measured: DAS28, CDAI, SDAI and ACR responses. Indeed, the equivalence margin of ±15% for the ACR20 rate difference, which was intended for the primary endpoint at week 30, was met also at week 54. Also, efficacy related to dose increments, whether regarding frequency or final dose, was comparable between SB2 and INF and is clinically consistent with results from the pivotal Safety Trial for Rheumatoid Arthritis with Remicade (infliximab) Therapy (START) study [30]. Thus, in a practical clinical setting where dose increments are allowed according to the instructions of the INF label, similar results can be expected with SB2. SB2 was well tolerated and demonstrated a comparable safety profile to INF. In general, the safety profile was comparable up to 54 weeks, with no particular difference from the 30 week report. The majority of TEAEs were considered to be mild to moderate in intensity and the incidence was comparable between the SB2 and INF treatment groups. There was no change in the incidence of alanine aminotransferase increases in the SB2 vs INF treatment groups compared with the 30 week report [16]. As seen from the 30 week results, our results continue to be comparable to other biosimilar RA studies of infliximab [31] up to 54 weeks, such as the rate of total and serious AEs. The incidence of ADA observed up to week 54 between SB2 and INF remained statistically non-significant and the trend was also comparable to what has been observed in the 30 week report. Any numerical difference did not result in a difference of efficacy or safety between the SB2 and INF treatment groups. Our results are considered comparable to other biosimilar RA studies [31]; it should be noted that our measure of ADA incidence is cumulative rather than at a single time point, resulting in a higher incidence than is seen in other such studies [31], also for reference INF. Indeed, as was seen in the 30 week results, these ADA incidences are higher than in the original INF pivotal studies [17], which is suggested to be due to the increased sensitivity of the assays. Our study has several strengths as an INF biosimilar study. As discussed previously, our study measured efficacy and safety at all visits. This allows a more sensitive assessment [32] and is considered to be close to proposed ‘standard’ designs in determining clinical biosimilarity [33]. Also, our study is the first among biosimilar infliximab studies to employ a dose increment scheme. While this design may have had the potential to introduce additional variability in efficacy responses after week 30, it is in line with clinical practice and the dose increments and efficacy response patterns remained comparable, further supporting biosimilarity. Our study also has some limitations. The study was not powered to detect a significant difference in radiographic progression between the treatment groups, thus drawing a definite conclusion regarding radiographic equivalence is not possible. Still, it is reassuring that the results were comparable on a numerical level, without any unexpected differences when compared with the efficacy or safety results. One of the major limitations of contemporary medical practice is the cost of medications, which has been cited as a major health policy goal [34]. bDMARDs are high-cost medications, and through our study we hope to contribute to a reduction of pressure on health care resources for bDMARD therapy [35]. BODY.CONCLUSION: SB2 demonstrated similar efficacy, safety and immunogenicity to its reference INF for up to 54 weeks in patients with moderate to severe RA despite MTX therapy. Such comparability was consistently maintained throughout the study and now includes the inhibition of radiographic progression. These data provide further evidence that SB2 is a biosimilar of INF. BODY.SUPPLEMENTARY MATERIAL: Supplementary Tables and FiguresClick here for additional data file.

TITLE: A clinical randomized trial on the use of atorvastatin in patients with sepsis or septic shock: effects on endothelial function BODY.INTRODUCTION: Sepsis and septic shock are complex inflammatory syndromes. Multiple cellular activation processes are involved, and many humoral cascades are triggered. Presumably, endothelial cells play a pivotal rule in the pathogenesis of sepsis, not only because they may influence the inflammatory cascade but also because, upon interaction with excessive amounts of inflammatory mediators, the function of these cells may become impaired. It is likely that a general dysfunction of the endothelium is a key event in the pathogenesis of sepsis [1]. HMG-CoA-reductase inhibitors have been shown to exhibit pronounced immunomodulatory effects independent of lipid lowering. Most of these beneficial effects of statins appear to involve restoring or improving endothelial function [2]. We hypothesize that statins can improve endothelial dysfunction in septic patients. BODY.METHODS: A double-blinded, placebo-controlled, randomized trial was undertaken. We enrolled adult patients within 24 hours of severe sepsis or septic shock diagnosis and randomized them to placebo or atorvastatin 80 mg/day for a short term. Endothelial dysfunction was assessed measuring plasmatic levels of IL-6, ET-1, VCAM-1 by ELISA and measuring flow-mediated vasodilatation of the brachial artery at basal, 24 and 72 hours after randomization. BODY.RESULTS: We studied 47 patients, 24 in the placebo group (mean age 52 ± 20 years, 29.1% male; APACHE II risk score 23.5 ± 7.3) and 23 in the statin group (mean age 49.5 ± 18 years, 53.4% male; APACHE II risk score 23 ± 6.9). The baseline characteristics of the placebo group were similar to statin patients as well as the mean length of stay in the ICU (8.6 ± 7.4 and 9.1 ± 8 days, respectively) and the time on vasopressors (49.3 ± 47.1 and 59 ± 91.1 hours, respectively). No significant difference was observed on the temporal variation of biomarker levels (IL-6, VCAM-1, ET-1) between treatment and control groups. The intrahospital mortality rate was 26% in the statin group and 45% in the placebo group (P = 0.17). BODY.CONCLUSION: Our data showed no benefit with the use of a potent statin acutely in patients with sepsis or septic shock with regards to improvement in endothelial function.

TITLE: Dipeptidyl Peptidase‐4 Inhibition Potentiates Stimulated Growth Hormone Secretion and Vasodilation in WomenWilson et al ABSTRACT.BACKGROUND: Diminished growth hormone (GH) is associated with impaired endothelial function and fibrinolysis. GH‐releasing hormone is the primary stimulus for GH secretion and a substrate of dipeptidyl peptidase‐4. We tested the hypothesis that dipeptidyl peptidase‐4 inhibition with sitagliptin increases stimulated GH secretion, vasodilation, and tissue plasminogen activator (tPA) activity. ABSTRACT.METHODS AND RESULTS: Healthy adults participated in a 2‐part double‐blind, randomized, placebo‐controlled, crossover study. First, 39 patients (29 women) received sitagliptin or placebo on each of 2 days separated by a washout. One hour after study drug, blood was sampled and then arginine (30 g IV) was given to stimulate GH. Vasodilation was assessed by plethysmography and blood sampled for 150 minutes. Following a washout, 19 of the original 29 women received sitagliptin alone versus sitagliptin plus antagonist to delineate GH receptor (GHR)– (n=5), nitric oxide– (n=7), or glucagon‐like peptide‐1 receptor– (n=7) dependent effects. Sitagliptin enhanced stimulated GH secretion (P<0.01 versus placebo, for 30 minutes) and free insulin–like growth factor‐1 (P<0.001 versus placebo, after adjustment for baseline) in women. Vasodilation and tPA increased in all patients, but sitagliptin enhanced vasodilation (P=0.01 versus placebo) and increased tPA (P<0.001) in women only. GHR blockade decreased free insulin–like growth factor‐1 (P=0.04 versus sitagliptin alone) and increased stimulated GH (P<0.01), but decreased vascular resistance (P=0.01) such that nadir vascular resistance correlated inversely with GH (r s=−0.90, P<0.001). GHR blockade suppressed tPA. Neither nitric oxide nor glucagon‐like peptide‐1 receptor blockade affected vasodilation or tPA. ABSTRACT.CONCLUSIONS: Sitagliptin enhances stimulated GH, vasodilation, and fibrinolysis in women. During sitagliptin, increases in free insulin–like growth factor‐1 and tPA occur via the GHR, whereas vasodilation correlates with GH but occurs through a GHR‐independent mechanism. ABSTRACT.CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01701973. BODY.CLINICAL PERSPECTIVE: BODY.WHAT IS NEW?: Dipeptidyl peptidase‐4 inhibition with the antidiabetic drug sitagliptin increases growth hormone (GH) secretion, vasodilation, and tissue plasminogen activator activity levels in women. During sitagliptin, increases in free insulin–like growth factor‐1 and tissue plasminogen activator activity occur through the GH receptor, whereas vasodilation correlates with GH but occurs independently of the GH receptor. BODY.WHAT ARE THE CLINICAL IMPLICATIONS?: These are the first data that suggest an off‐target effect of the antidiabetic drug sitagliptin on endogenous GH secretion. Our findings suggest that one mechanism by which sitagliptin affects the vasculature is by enhancing GH secretion. Dipeptidyl peptidase‐4 inhibition may be a novel pharmacologic mechanism to enhance endogenous GH and insulin‐like growth factor‐1 secretion and thereby mitigate cardiovascular risk in patients with attenuated GH secretion. BODY.INTRODUCTION: The growth hormone (GH) insulin‐like growth factor‐1 (IGF‐1) axis regulates vascular function and fibrinolytic capacity in adults. GH is secreted in a pulsatile fashion from the pituitary gland and acts directly or indirectly through IGF‐1, which is secreted by the liver in response to hepatic GH receptor (GHR) activation. GH and IGF‐1 receptors are ubiquitously present on human endothelial cells and the myocardium.1, 2 In healthy patients, acute systemic and intra‐arterial GH infusion increases GH without affecting IGF‐1, and increases endothelium‐dependent vasodilator function.3, 4 Patients with GH deficiency demonstrate altered fibrinolytic balance, characterized by elevated plasminogen activator inhibitor‐1 (PAI‐1) antigen levels and lower tissue plasminogen activator (tPA) activity,5 which resolves with GH replacement therapy.6 IGF‐1 also exerts many favorable endothelial effects via the IGF‐1 receptor, including enhanced endothelium‐dependent vasodilation, anti‐inflammatory effects, and maintenance of vascular integrity via activated endothelial progenitor cells.2, 7 Thus, the vascular effects of GH may be mediated by either direct effects of GH or indirect effects of increased IGF‐1. Adults with low GH or IGF‐1 levels have elevated cardiovascular risk. Patients who have attenuated GH secretion include those with abdominal obesity, physiologic aging, HIV infection, polycystic ovarian syndrome, and pituitary and hypothalamic disease.8, 9, 10 In cardiovascular epidemiology studies, individuals in the lowest IGF‐1 quartile have a higher risk of ischemic heart disease,11 heart failure,12 and all‐cause mortality.13 Similarly, overweight and obese adults demonstrate a decreased response to GH secretagogues, which correlates with the presence of cardiovascular risk markers.14 For this reason, pharmacologic interventions designed to increase GH and IGF‐1 in deficient populations have been developed in the past 2 decades in hopes of mitigating cardiovascular risk. Recombinant GH therapy was the first such therapy approved to increase GH and IGF‐1 levels in deficient patients; however, exogenous GH is not restrained by physiologic negative feedback by IGF‐1, does not restore pulsatile secretion, and is limited by the side effect of hyperglycemia. An alternative and previously unexplored method to enhance GH and downstream IGF‐1 secretion in humans is to inhibit the degradation of endogenous GH‐releasing hormone (GHRH) by dipeptidyl peptidase‐4 (DPP4). GHRH is the primary stimulus for pituitary GH secretion and determines GH pulsatility. Endogenous GHRH has a half‐life of ≈6 minutes in humans as it is degraded and inactivated by DPP4.15 Sitagliptin was the first DPP4 inhibitor approved by the US Food and Drug Administration in 2006 for the management of hyperglycemia in patients with type 2 diabetes mellitus. Sitagliptin decreases the degradation of the incretin hormone, glucagon‐like peptide‐1 (GLP‐1), and thereby improves postprandial hyperglycemia in patients with diabetes mellitus in a glucose‐dependent manner. In this study, we tested the hypothesis that inhibition of DPP4 activity by sitagliptin would enhance stimulated GH secretion and thereby increase vasodilation and plasma tPA activity in healthy young men and women. To further elucidate the mechanism by which stimulating the GH–IGF‐1 axis influences vascular function and fibrinolysis, we then investigated the contribution of nitric oxide, GHR activation, and GLP‐1 receptor activation to the vascular changes observed with stimulated GH secretion during DPP4 inhibition. BODY.METHODS: Anonymized data and analytic methods will be made publicly available (NCT01701973 at www.clinicaltrials.gov). BODY.STUDY PROTOCOL: Healthy, lean (body mass index ≤25 kg/m2), nonsmoking adults, aged 18 to 40 years, participated in a 2‐part double‐blind, randomized, placebo‐controlled, crossover study. (See Table 1 for patient characteristics.) The study adhered to the principles of the Declaration of Helsinki and Title 45, US Code of Federal Regulations, Part 46, Protection of Human Subjects, and was approved by the Vanderbilt University Medical Center's institutional review board. All patients provided written informed consent before initiation of study procedures. Patients with a history of chronic illness, including diabetes mellitus, hypertension, cardiovascular disease, and chronic renal or hepatic insufficiency, were excluded. Medication use other than a multivitamin was prohibited at the time of study; oral contraceptive use was not permitted in women. Pregnancy was excluded in women of child‐bearing age by serum pregnancy testing. Table 1Patient Characteristics ParameterN=39Age, y 25±5 Race, No. (%)  White 31 (80)  Black 2 (5)  Hispanic 4 (10)  Asian 2 (5) Sex, No. (%)  Women 29 (74)  Men 10 (26) Weight, kg 65.4±9.3 Body mass index, kg/m2 22.9±1.8 Values are expressed as mean±SD unless otherwise indicated. In the first half of the study (Figure S1A), 39 patients underwent 2 study days separated by a washout period to determine the effect of DPP4 inhibition on stimulated GH secretion. Patients were assigned to treatment order (sitagliptin or matching placebo) using a block randomization algorithm. On each study day, patients reported to the Vanderbilt Clinical Research Center in the morning after an overnight fast. All patients were studied in the supine position in a temperature‐controlled room. Participants were given oral study drug (sitagliptin or matching placebo), and a peripheral intravenous line was placed in the antecubital fossa of their nondominant arm. Sitagliptin dose was 200 mg in the first 14 patients (7 women) to achieve >80% inhibition of DPP4 activity within 1 hour. We changed to sitagliptin 100 mg daily for 4 days in the remaining 25 patients to achieve steady‐state dosing and to be consistent with the current Food and Drug Administration–approved dose. Study days were separated by a 1‐week washout for the first 14 participants and a 2‐week washout in the remaining 25 participants. Vasodilation was assessed via strain‐gauge plethysmography (Data S1) and venous blood samples were obtained 60 minutes following study drug. Patients then received arginine (30 g) intravenously over 30 minutes to stimulate endogenous GH secretion in a GHRH‐dependent manner, as previously described.16 Vasodilation was assessed and venous blood samples were obtained for 150 minutes after completion of arginine infusion. On the second study day, the protocol was repeated using the opposite study drug (sitagliptin or matching placebo). Blood pressure and heart rate were monitored throughout each study day. Following at least an 8‐week washout period, 19 of the 29 women from the first half of the study participated in 2 additional study visits designed to elucidate the mechanism by which sitagliptin potentiated vasodilation and tPA release (Figure S1B). We studied women only based on the results of the first part of the study. Each patient's sitagliptin dose and washout period was the same as in the first half of the protocol. Patients were divided into 3 subgroups. In the first subgroup, 5 women were randomized to a single double‐blinded subcutaneous injection of saline vehicle or pegvisomant 80 mg (Pfizer Inc) administered 72 hours before the study day to block the GHR. This dose of pegvisomant produces peak drug levels 72 hours following administration with a reduction in free IGF‐1 indicative of efficient GHR blockade.17 In the second subgroup, 7 women were randomized to either double‐blinded saline vehicle infusion or the nitric oxide synthase inhibitor, l‐N‐monomethylarginine (LNMMA [acetate]; Clinalfa; Bachem Americas, Inc), administered as a 3‐mg/kg 15‐minute intravenous infusion preceding arginine followed by an additional 6 mg/kg infused over 120 minutes. This dose demonstrates peak hemodynamic effects 20 minutes after the start of the infusion and does not affect arginine‐stimulated GH secretion.18, 19 In the third subgroup, 7 women were randomized to either double‐blinded saline vehicle infusion or Exendin 9‐39 (Exendin 9‐39 Acetate; Clinalfa; Bachem Americas, Inc), administered as an intravenous bolus infusion of 7500 pmol/kg over 1 minute preceding arginine followed by a continuous infusion of 750 pmol/kg per minute for 150 minutes, to block the GLP‐1 receptor.20 BODY.LABORATORY ANALYSES: All samples were obtained after the first 3 mL of blood were discarded. Blood samples were collected on ice, centrifuged immediately, and plasma‐stored at −80°C in prespecified aliquots until time of assay. Venous DPP4 antigen concentration was determined by ELISA (eBioscience). Venous DPP4 activity was assayed by incubating 20 μL of serum sample in 80 μL assay buffer (0.1 mol/L Tris at a pH of 8.0; Bachem) for 30 minutes at 37°C with colorimetric substrate (2 mmol/L L‐glycyl‐L‐prolyl p‐nitroanilide hydrochloride [Sigma Aldrich]) for a total reaction volume of 200 μL, as previously described.21 The enzyme activity was assessed by measuring the increase in specific absorbance at 405 nm at 0, 15, and 30 minutes and was expressed as nmol/mL per minute. GH levels were determined using the Access Ultrasensitive hGH Assay (Beckman Coulter), while GH levels after pegvisomant administration were analyzed using the IDS‐iSYS hGH assay; both assays were calibrated against National Institute for Biological Standards and Control World Health Organization International Standard 98/574. Free IGF‐1 was determined using a commercially available ELISA (R&D systems). IGF‐1 was analyzed by Luminex assay (EMD Millipore), which is calibrated against the National Institute for Biological Standards and Control World Health Organization International Standard 02/254. tPA activity and PAI‐1 antigen levels were measured in blood collected in acidified citrate anticoagulant (TriniLIZE Stabilyte tubes, Tcoag; Bray Co). tPA activity was analyzed using a biofunctional immunosorbent assay calibrated against National Institute for Biological Standards and Control World Health Organization International Standard 86/670 (TriniLIZE tPA Activity, Tcoag, Co) in the first 14 patients. Following discontinuation of this assay, the remaining samples were analyzed using an ELISA calibrated against National Institute for Biological Standards and Control World Health Organization International Standard 98/714 (Oxford Biomedical Research). Samples from each patient were assayed using the same method. PAI‐1 antigen was analyzed using a TintElize PAI‐1 antigen assay (Tcoag, Co). Plasma cGMP was determined using a competitive enzyme immunoassay (GE Healthcare Bio‐Sciences Corp). Samples for analysis of active GLP‐1 were collected in aprotinin and analyzed using the MILLEPLEX MAP Human Metabolic Hormone Magnetic Bead Panel (EMD Millipore Corporation). Insulin and estradiol were analyzed by double‐antibody radioimmunoassay. Bedside blood glucose was determined by YSI bedside glucose analyzer (YSI Life Sciences). BODY.STATISTICAL ANALYSIS: Data are presented as mean±SD, unless otherwise noted. We tested for carryover effect using the t test approach proposed by Jones and Kenward.22 Wilcoxon signed‐rank test was used to compare baseline variables between treatment conditions as well as GH levels (untransformed) between treatment conditions at each time point. Wilcoxon rank‐sum test was used to compare percent DPP4 inhibition before GH stimulation, peak GH during placebo, and peak GH during sitagliptin between men and women. Percent DPP4 inhibition was determined by the equation: [1−(DPP4 activity during sitagliptin/DPP4 activity during placebo)]×100. Spearman correlation was used to evaluate the association between continuous variables. Mixed effect models were used to analyze the data with a random subject effect and with fixed effects of treatment (sitagliptin versus placebo or sitagliptin+antagonist versus sitagliptin+placebo), time, and treatment×time interaction. The baseline measurement was also included in each model. Interaction terms were removed from the final model when the P value from the corresponding overall test for interaction was >0.2. Results from mixed effect models are presented as the mean difference between treatments with 95% confidence interval. The end points GLP‐1, insulin, and GH were log transformed to satisfy model assumptions. Statistical analyses were performed using IBM SPSS software version 23.0, GraphPad Prism 5 and R 2.15.0 (www.r-project.org). Sample size calculations are included in Data S1. BODY.RESULTS: BODY.EFFECT OF SITAGLIPTIN ON DPP4 ACTIVITY AND GLP‐1: Sitagliptin significantly decreased DPP4 activity (P<0.001 versus placebo) and increased GLP‐1 levels both at baseline and throughout stimulated GH secretion (P<0.0001 versus placebo) (Table 2). Sitagliptin did not affect insulin levels (P=0.45 versus placebo) or blood glucose levels (P=0.58 versus placebo) during stimulated GH secretion or at baseline (Table 2). Sitagliptin (200 mg) reduced DPP4 activity similarly in men and women (percent DPP4 inhibition 78±13% in 7 women versus 80±6% in 7 men, P>0.999). Safety data are summarized in Data S1. Table 2Initial Biochemical Parameters Before GH Stimulation VariablePlaceboSitagliptinNa P ValueDPP4 activity, nmol/mL per minute 25.4±6.5 (24.8, 9.7) 8.1±4.5 (7.0, 5.6) 39 <0.001 DPP4 antigen, ng/mL 458.1±158.1 (458.4, 191.7) 431.2±149.4 (423.8, 250.7) 38 0.22 Blood glucose, mg/dL 86.6±5.8 (86.0, 8.4) 85.4±7.8 (85.1, 7.9) 39 0.24 Insulin, μU/mL 6.8±3.2 (6.6, 4.1) 7.3±4.0 (7.0, 6.0) 39 0.30 GLP‐1, pg/mL 2.7±2.8 (2.0, 0.8) 19.1±12.5 (19.1, 16.6) 39 <0.001 Estradiol, pg/mLb 252.9±256.6 (177.6, 145.7) 250.9±173.9 (238.4, 195.8) 29 0.67 tPA activity, IU/mL 0.19±0.18 (0.14, 0.18) 0.24±0.26 (0.14, 0.31) 36 0.13 PAI‐1 antigen, ng/mL 4.4±4.1 (3.0, 4.6) 3.5±2.7 (2.9, 4.0) 36 0.26 Total IGF‐1, ng/mL 108.3±30.4 (102.3, 46.6) 104.6±30.5 (106.8, 38.6) 39 0.11 Free IGF‐1, ng/mL 0.65±0.31 (0.61, 0.45) 0.65±0.24 (0.66, 0.39) 37 0.67 Results are presented as mean±SD (median, interquartile range). DPP4 indicates dipeptidyl peptidase‐4; GLP‐1, glucagon‐like peptide‐1; IGF‐1, insulin‐like growth factor‐1; PAI‐1, plasminogen activator inhibitor‐1; tPA, tissue plasminogen activator. aResults were analyzed in patients with data available on both days. bAnalyzed in women only. BODY.EFFECT OF SITAGLIPTIN ON STIMULATED GH SECRETION AND FREE IGF‐1: Arginine infusion stimulated GH secretion to a greater extent in women (n=29) than in men (n=10), as previously described,23 during both placebo (peak GH 9.8±5.0 ng/mL women versus 5.7±3.2 ng/mL men, P=0.02) and sitagliptin (11.6±5.7 ng/mL women versus 5.1±4.6 ng/mL men, P<0.01). Sitagliptin significantly enhanced GH secretion following arginine infusion in women (P=0.01 versus placebo at arginine completion, P=0.02 versus placebo 15 minutes after arginine, and P=0.09 versus placebo 30 minutes after arginine) but not men (P=0.49 versus placebo at arginine completion, P=0.77 versus placebo 15 minutes after arginine, and P=0.70 versus placebo 30 minutes after arginine) (Figure 1A). The effect of treatment on ln(GH), as determined by linear model, was significant in women for 30 minutes following arginine (P<0.01 versus placebo, after adjustment for baseline GH). Similarly, sitagliptin increased free IGF‐1 levels during stimulated GH secretion in women (P<0.001 versus placebo, after adjustment for baseline free IGF‐1) but not in men (P=0.39 versus placebo, after adjustment for baseline free IGF‐1). Figure 1B shows change in free IGF‐1 from baseline to 90 minutes after arginine. Sitagliptin also shortened the time to peak GH in women (P<0.01 versus placebo) but not men (P=0.62 versus placebo) (Figure 1C). Figure 1Dipeptidyl peptidase‐4 (DPP4) inhibition with sitagliptin enhances early stimulated growth hormone (GH) secretion (A) and free insulin–like growth factor‐1 (IGF‐1) levels (B) and shortens the time to peak GH (C) in women (n=29) but not men (n=10). Change in free IGF‐1 levels from baseline to 90 minutes following arginine. (Free IGF‐1 data available in 28 women and 9 men.) Data are presented as mean±SEM. *P≤0.05; † P<0.10 vs placebo at same time point by Wilcoxon signed‐rank test. The effect of treatment on free IGF‐1, in the linear model, was significant in women (P<0.001 vs placebo, after adjustment for baseline free IGF‐1). The effect of treatment on ln(GH), in the linear model, was significant in women for 30 minutes following arginine (P<0.01 vs placebo, after adjustment for baseline GH). BODY.EFFECT OF SITAGLIPTIN ON VASODILATION AND CGMP LEVELS DURING STIMULATED GH SECRETION: DPP4 inhibition did not significantly affect blood pressure, pulse rate, or vasodilation before arginine infusion, as compared with placebo (Table 3). Vasodilator response is presented as the change in both forearm blood flow (FBF) and forearm vascular resistance (FVR) following arginine infusion. FBF increased (P<0.001 effect of time) and FVR decreased (P<0.001 effect of time) following stimulated GH secretion. Sitagliptin enhanced the increase in FBF (P=0.01 versus placebo) and decrease in FVR (P=0.003 versus placebo) in women only (Figure 2). Sitagliptin also increased pulse rate (P=0.03 versus placebo) following stimulated GH secretion in women. Sitagliptin increased cGMP levels during stimulated GH secretion in both women (increase of 98.57 fmol [95% confidence interval, 33.18–163.96], P=0.003 versus placebo) and men (increase of 116.39 fmol [95% confidence interval, 43.33–189.33], P=0.002 versus placebo). Table 3Initial Hemodynamic Parameters Before GH Stimulation VariablePlaceboSitagliptin P ValueSystolic BP, mm Hg 108.8±10.8 (110.0, 18.0) 109.9±10.7 (108.0, 12.0) 0.40 Diastolic BP, mm Hg 65.6±6.4 (65.0, 9.0) 65.6±6.5 (66.0, 6.0) 0.97 Mean arterial pressure, mm Hg 82.9±6.2 (84.0, 10.0) 83.2±5.9 (83.0, 6.0) 0.59 Pulse rate, beats per min 58.3±8.3 (57.0, 13.0) 59.3±8.1 (60.0, 11.0) 0.29 FVR, mm Hg/(mL/min/100mL) 36.9±11.4 (36.2, 19.4) 37.4±10.2 (35.6, 12.3) 0.58 FBF, mL/min per 100 mL 2.5±0.9 (2.2, 1.3) 2.4±0.6 (2.4, 0.8) 0.65 BP indicates blood pressure; FBF, forearm blood flow; FVR, forearm vascular resistance; GH, growth hormone. Results are presented as mean±SD (median, interquartile range). Figure 2Dipeptidyl peptidase‐4 inhibition with sitagliptin enhances vasodilation during arginine (Arg) stimulated growth hormone secretion in women (n=29) but not men (n=10). The overall effect of treatment, as determined by linear model, was significant in women (P=0.013 effect of treatment on percent change [∆] in forearm blood flow and P=0.003 effect of treatment on percent change in forearm vascular resistance). Data are presented as mean±SEM. *P<0.05 vs placebo at specified time point in the linear model. BODY.EFFECT OF SITAGLIPTIN ON TPA ACTIVITY DURING STIMULATED GH SECRETION: tPA activity increased following stimulated GH secretion (P<0.001). Acute inhibition of DPP4 activity with 200 mg of sitagliptin increased tPA activity levels in women (P<0.001 versus placebo, n=7) but decreased tPA activity in men (P=0.02 versus placebo, n=7) (Figure 3). This effect of DPP4 inhibition on tPA activity levels was not observed following 100 mg of sitagliptin. PAI‐1 antigen levels during stimulated GH secretion were unaffected by sitagliptin in men and women (P=0.33 versus placebo). Figure 3Dipeptidyl peptidase‐4 inhibition with 200 mg sitagliptin increases tissue plasminogen activator (tPA) activity levels in women (n=7 women) but decreases tPA activity levels in men relative to baseline (n=7 men) during arginine stimulated growth hormone secretion. The overall effect of treatment on tPA activity, as determined by linear model, was significant after adjustment for baseline tPA activity (P<0.001 effect of treatment in women and P=0.02 effect of treatment in men). There was no effect of 100 mg daily of sitagliptin on tPA activity. Data are presented as mean±SEM. *P<0.05 vs placebo at specified time point in linear model after adjustment for baseline tPA. BODY.EFFECT OF GHR BLOCKADE ON VASODILATION AND TPA ACTIVITY DURING STIMULATED GH SECRETION IN WOMEN: Pegvisomant significantly decreased free IGF‐1 during sitagliptin (P=0.04 versus sitagliptin alone, n=5) and increased GH levels (P<0.01 versus sitagliptin alone), consistent with effective GHR blockade (Figure 4A). The addition of GHR blockade significantly increased vasodilation (P<0.01 versus sitagliptin alone for change in FVR) throughout stimulated GH secretion. Moreover, at the nadir in vascular resistance, GH levels correlated inversely with vascular resistance (r s=−0.90, P<0.001) (Figure 4B). Pegvisomant suppressed tPA activity before (0.24±0.12 after sitagliptin alone versus 0.10±0.08 IU/mL after addition of pegvisomant, P=0.04) and during stimulated GH secretion (P<0.001 versus sitagliptin alone) (Figure 4C). Figure 4The addition of growth hormone (GH) receptor blockade (pegvisomant 80 mg administered SC 72 hours prior) to sitagliptin increases GH levels as a result of reduced negative feedback (n=5 women) (A) and decreases free insulin–like growth factor‐1 (IGF‐1) (B). The addition of GH receptor blockade to sitagliptin further decreases forearm vascular resistance (FVR) during stimulated GH secretion (C). At the nadir in vascular resistance (arrow), a significant correlation between vascular resistance and GH levels was found (D). Pegvisomant decreases tissue plasminogen activator (tPA) activity levels before and throughout arginine (Arg)‐stimulated GH secretion (E). Data are presented as mean±SEM. *P≤0.05 vs placebo at specified time point in the linear model. Linear model–based P values are: P<0.01 effect of treatment on GH, P<0.01 effect of treatment on FVR percent change (∆), and P<0.001 effect of treatment on tPA activity. BODY.EFFECT OF NO SYNTHASE INHIBITION ON VASODILATION AND TPA ACTIVITY DURING STIMULATED GH SECRETION IN WOMEN: LNMMA significantly decreased cGMP levels during sitagliptin and stimulated GH secretion (decrease of 432.39 fmol [95% confidence interval, −792.77 to −72.02], P=0.02 versus sitagliptin alone, n=7). LNMMA did not affect the vasodilator response to stimulated GH secretion during sitagliptin (P=0.43 versus sitagliptin alone for change in FBF and P=0.94 versus sitagliptin alone for change in FVR) (Figure 5A). The addition of LNMMA to sitagliptin also had no effect on tPA activity (P=0.64 versus sitagliptin alone) (data not shown). Figure 5 l‐N‐monomethylarginine (LNMMA) did not affect the vasodilator response to arginine (Arg)‐stimulated growth hormone (GH) secretion during sitagliptin (A) (n=7 women). Glucagon‐like peptidase‐1 (GLP‐1) receptor blockade (Exendin 9‐39) did not affect the vasodilator response to stimulated GH secretion during sitagliptin (B) (n=7 women). Data are presented as mean±SEM. *P<0.05 vs placebo at specified time point in the linear model. The linear model–based P values for overall effect of treatment were not significant. BODY.EFFECT OF GLP‐1 RECEPTOR BLOCKADE ON VASODILATION AND TPA ACTIVITY DURING STIMULATED GH SECRETION IN WOMEN: GLP‐1 receptor blockade with Exendin 9‐39 increased fasting GLP‐1 (P<0.01), glucagon (P=0.09), and blood glucose levels (P<0.001), as previously described.20, 24, 25 Exendin 9‐39 briefly caused vasoconstriction immediately after arginine infusion (P=0.02 versus sitagliptin alone for FBF and P=0.02 versus sitagliptin alone for FVR at 60 minutes, n=7) (Figure 5B). Following stimulated GH secretion, FBF increased (P<0.001 effect of time) and FVR decreased (P<0.001 effect of time). The addition of Exendin 9‐39 to sitagliptin did not prevent vasodilation following stimulated GH secretion (P=0.88 versus sitagliptin alone for change in FBF and P=0.57 versus sitagliptin alone for change in FVR). The addition of Exendin 9‐39 to sitagliptin also had no effect on tPA activity (P=0.58 versus sitagliptin alone) (data not shown). BODY.REPRODUCIBILITY OF STIMULATED GH SECRETION DURING DPP4 INHIBITION: The reproducibility of the effect of DPP4 inhibition on stimulated GH secretion was assessed by comparing GH levels during sitagliptin alone with GH levels obtained during sitagliptin plus saline vehicle infusion in the 19 women who completed both crossover studies (Figure 6). There was a significant correlation between stimulated GH secretion following sitagliptin and stimulated GH secretion following sitagliptin plus saline infusion (peak GH response: r s=0.65, P=0.003; GH 30 minutes after arginine: r s=0.51, P=0.02). Figure 6The increase in arginine (Arg)‐stimulated growth hormone (GH) secretion during dipeptidyl peptidase‐4 inhibition with sitagliptin is reproducible (n=19 women). Data are presented as mean±SEM unless otherwise noted. There was a significant correlation between stimulated GH secretion following sitagliptin and stimulated GH secretion following sitagliptin plus saline infusion (peak GH response: r s=0.65, P=0.003; GH 30 minutes after arginine: r s=0.51, P=0.02). BODY.DISCUSSION: This study tested the hypothesis that DPP4 inhibition potentiates arginine‐stimulated GH secretion in humans. We found that sitagliptin significantly enhanced stimulated GH secretion and shortened the time to peak GH in healthy women but not men. Similarly, sitagliptin increased free IGF‐1 levels in women. Forearm vasodilation after peak GH was potentiated by sitagliptin only in women. GHR blockade further increased vasodilation during DPP4 inhibition in association with increased GH levels. The latter indicates that GH induces endothelium‐independent vasodilation through a GHR‐independent mechanism. Our study is the first to define an off‐target effect of the antidiabetic medication sitagliptin on GH and the first study of the effect of DPP4 inhibition on the GH axis to include women. An understanding of the effect of DPP4 inhibition on GH can only be achieved by studying humans because of significant interspecies variation in the neuroregulation of GH secretion.26 Bergman et al27 examined the effect of 10‐day treatment with sitagliptin, in doses ranging from 25 mg daily to 300 mg twice daily, on IGF‐1 levels in 8 healthy young men. Although IGF‐1 increased in nearly every treatment group after 10 days, the change was not statistically significant. Schopman et al28 reported that sitagliptin decreased GH levels after insulin‐induced hypoglycemia in insulin‐dependent men with diabetes mellitus and attributed this to an inhibitory effect of GLP‐1 stimulation. GLP‐1 is unlikely to contribute to enhanced GH secretion in women in the present study, as GLP‐1 receptor blockade did not influence GH secretion. We found an effect of sitagliptin in women but not in men, consistent with the mechanism of known sexual dimorphism in GH secretion. The observation that sitagliptin affects GH secretion in women only may be explained by work of others, which demonstrates that GHRH drives GH pulsatility in women and that interpulse GH secretion in women is more GHRH dependent and less susceptible to feedback inhibition by free IGF‐1.29, 30, 31 We also observed that arginine did not stimulate GH secretion as effectively in men. This dose of arginine may have been insufficient for us to detect a significant effect of sitagliptin on stimulated GH secretion in men. Given these known differences in GH secretion between men and women, a separate adequately powered study is needed to investigate what effect sitagliptin has on GH secretion in men. Our study is unique in that it investigates for the first time in humans the specific contribution of GHR activation and IGF‐1 to the vasodilation observed after an increase in GH. The vasodilator effects of GH and IGF‐1 in humans are well described. Napoli et al4 demonstrated that intra‐arterial GH resulting in a 10‐fold increase in GH levels in the human forearm with no effect on IGF‐1 doubles forearm blood flow several hours later. IGF‐1 causes relaxation in harvested human internal mammary artery that is unaffected by removal of the endothelium and NO inhibition but abolished by potassium chloride, suggesting that IGF‐1–induced vasorelaxation in humans involves the potassium channels in vascular smooth muscle cells.32 We are not aware of any studies evaluating the effect of IGF‐1 on the vasculature in the human forearm. We found that GHR blockade paradoxically increased vasodilation following arginine‐stimulated GH release in sitagliptin‐treated women, even though IGF‐1 concentrations were decreased. Increased vasodilation correlated with GH concentrations, which were increased as a result of loss of feedback inhibition, consistent with a GHR‐ and IGF‐1–independent vasodilator effect of GH. Consistent with this mechanism, chronic pegvisomant therapy in patients with acromegaly improves flow‐mediated dilation.33 GH treatment in hypophysectomized rats upregulates vascular smooth muscle ATP‐sensitive potassium channel mRNA,34 which could lead to depolarization and vasorelaxation. Lastly, adults with primary GH resistance, characterized by elevated GH but defective GHR signaling and IGF‐1 deficiency, demonstrate normal conduit artery vascular function.35 Late vasodilation after arginine is believed to be mediated by GH, as octreotide infusion blocks arginine‐stimulated GH secretion and prevents vasodilation.36 GH has also been reported to cause vasodilation via NO‐dependent mechanisms. Prior studies indicate that coinfusion of LNMMA decreases the vasodilator response to GH.4 Li et al3 corroborated these findings using an acute systemic GH infusion and further demonstrated that GH increases phosphorylation and activity of endothelial NO synthase in human aortic endothelial cells in vitro. Others have found that chronic GH therapy, which also increases IGF‐1 levels, increases markers of NO bioavailability, decreases peripheral resistance, and improves conduit artery vascular function.37, 38 Our results do not support a contribution by NO to enhanced vasodilation following arginine‐stimulated GH secretion during DPP4 inhibition, as vasodilation was not blocked by LNMMA and thus was NO synthase independent. Sitagliptin also increases levels of intact GLP‐1 and insulin, and intact GLP‐1 has previously been reported to enhance the vasodilator response to intra‐arterial insulin in adults with metabolic syndrome.39 It is unlikely that increased insulin and GLP‐1 receptor activation contributed to the enhanced vasodilation during sitagliptin. Insulin levels were identical across study days and the mechanism of vasodilation was NO synthase independent. While sitagliptin increased fasting GLP‐1 levels, the addition of GLP‐1 receptor blockade did not prevent vasodilation. Furthermore, our group previously demonstrated that intra‐arterial infusion of GLP‐1 in the setting of sitagliptin has no effect on vasodilation in the forearm of healthy adults.40 Ban et al41 demonstrated that GLP‐1 (9‐36) increases endothelium‐dependent vasodilation in mice lacking a GLP‐1 receptor. A GLP‐1 receptor‐independent mechanism involving GLP‐ We observed an increase in tPA activity during stimulated GH secretion that was potentiated by sitagliptin in women. We previously demonstrated that adults with GH deficiency have decreased tPA activity, along with a defective fibrinolytic response to venous occlusion.5 Miljic et al6 also reported an improvement in stimulated endothelial tPA release following venous occlusion after 1 year of GH replacement aimed to normalize IGF‐1 in GH‐deficient adults. We demonstrated that GH mediates tPA activity levels through the GHR in women, as the addition of GHR blockade suppressed tPA activity before and during stimulated GH secretion. Muller et al42 found no effect of pegvisomant on tPA activity in men, whereas we detected a decrease in tPA activity. BODY.STUDY LIMITATIONS AND STRENGTHS: Our findings are limited by a few study design considerations. We studied healthy, lean individuals to avoid medications and diseases that may affect stimulated GH secretion, endothelial function, and fibrinolysis. We studied patients who fasted, while sitagliptin exerts its incretin‐specific effects in the postprandial state. Because sitagliptin may influence GH secretion by lowering postprandial blood sugar and free fatty acids, sitagliptin could have a greater effect on GH secretion in the postprandial state. We administered sitagliptin for a limited duration to decrease the likelihood of an increase in IGF‐1 influencing GH secretion through feedback inhibition. The effect of chronic sitagliptin on GH secretion may be less pronounced. Measurement of plasma GHRH levels was not performed, given the several 1000‐fold dilution of pituitary‐portal GHRH and the inability of assays to distinguish between pituitary and peripheral GHRH. We used arginine as a stimulus for endogenous GH secretion. Other currently available GH secretagogues, insulin and glucagon, may be unsafe to administer during sitagliptin and would cause hormonal changes that would confound results. While arginine‐stimulated GH secretion is unaffected by phase of the menstrual cycle,43 vasodilation is highest during the late follicular phase when estradiol levels are highest.44 We were limited in our ability to coordinate all assessments to the same phase of the menstrual cycle in our participants; however, we did not observe a difference in estradiol levels across study treatments. Similar to other researchers, we found low intraindividual variability in the GH response to arginine.45 In fact, a strength of our study is the highly reproducible stimulated GH secretion profile during sitagliptin in women. We were not able to confirm complete blockade of the GHR. The resulting increased GH levels may have been sufficient to stimulate an incompletely blocked GHR albeit to a lesser degree. Our study is also limited by our inability to define the mechanism by which increased GH causes sex‐specific vasodilation. Further study using glibenclamide in an appropriate study population may elucidate a contribution by the vascular smooth muscle ATP‐sensitive potassium channel. Lastly, our limited sample size in the second half of the study limits our conclusions on the impact of NO synthase inhibition and GLP‐1 receptor blockade. BODY.CONCLUSIONS: In this study, we tested a previously unexplored method to enhance endogenous pulsatile GH secretion in humans using the oral antidiabetic DPP4 inhibitor sitagliptin. Our data are the first to support an off‐target effect of DPP4 inhibition on GH secretion in women. This is clinically relevant for 2 reasons. First, the cardiovascular effects of DPP4 inhibition and how these differ from the effects of GLP‐1 analogues is an active area of investigation. Our findings suggest that DPP4 inhibition influences vasodilation by influencing GH secretion in women. Second, while current strategies to increase GH cause hyperglycemia, oral DPP4 inhibitor therapy offers an attractive, novel mechanism to enhance endogenous GH secretion while also improving glucose metabolism. It was previously well established that increases in GH and IGF‐1 improve fibrinolytic capacity, vasodilator function, and lower inflammation. Medications that potentiate GH secretion may thus prevent atherosclerosis through these mechanisms. Last, an enhanced understanding of the specific effects of GH, GHR activation, and IGF‐1 on cardiovascular risk and how they are modified by sex is critical to how we incorporate somatotropic medications in the management algorithms for patients with impaired GH secretion and elevated cardiovascular risk. BODY.SOURCES OF FUNDING: This research was supported by Vanderbilt Clinical and Translational Science Awards (CTSA) grant UL1 TR000445‐06 from the National Institutes of Health National Center for Advancing Translational Sciences. The Vanderbilt Hormone Assay and Analytical Services Core is supported by National Institutes of Health grants DK059637 and DK020593. Devin was supported by K23HL11962 National Heart, Lung, and Blood Institute/National Institutes of Health, Brown by R01HL125426 from National Heart, Lung, and Blood Institute/National Institutes of Health and 17SFRN33520017 from the American Heart Association, and Wilson by T32GM007569 from National Institute of General Medical Sciences/National Institutes of Health and T32DK007061 from National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health. BODY.DISCLOSURES: None. BODY.SUPPORTING INFORMATION: Data S1. Supplemental methods. Figure S1. Patients participated in a 2‐part double‐blind, randomized, placebo‐controlled, crossover study. Click here for additional data file.

TITLE: Effects of Ulinastatin on Postoperative Blood Loss and Hemostasis in Atrioventricular Valve Surgery with Cardiopulmonary Bypass ABSTRACT.BACKGROUND: Cardiopulmonary bypass (CPB) induces variable systemic inflammatory reactions associated with major organ dysfunction via polymorphonuclear neutrophils (PMNs). Ulinastatin, a urinary trypsin inhibitor, inhibits PMN activity and reduces systemic inflammatory responses. The aim of this study is to evaluate the effect of ulinastatin on postoperative blood loss and laboratory changes in patients undergoing open heart surgery. ABSTRACT.MATERIALS AND METHODS: Between January 2008 and February 2009, 110 patients who underwent atrioventricular valve surgery through right thoracotomy were divided into two groups. Patients received either 5,000 U/kg ulinastatin (ulinastatin group, n=41) or the equivalent volume of normal saline (control group, n=69) before aortic cross clamping. The primary end points were early coagulation profile changes, postoperative blood loss, transfusion requirements, and duration of intubation and intensive care unit stay. ABSTRACT.RESULTS: There were no statistically significant differences between the two groups in early coagulation profile, other perioperative laboratory data, and postoperative blood loss with transfusion requirements. ABSTRACT.CONCLUSION: Administration of ulinastatin during operation did not improve the early coagulation profile, postoperative blood loss, or transfusion requirements of patients undergoing open heart surgery. In addition, no significant effect of ulinastatin was observed in major organs dysfunction, systemic inflammatory reactions, or other postoperative profiles. BODY.INTRODUCTION: Open heart surgery using cardiopulmonary bypass (CPB) with aortic cross-clamping (ACC) provokes various systemic inflammatory responses that may eventually lead to multiple-organ injury or dysfunction. Such systemic reactions are characterized by an activation of pro-inflammatory cytokines, protease enzymes, and oxygen free radicals from activated neutrophils resulting in endothelial injury, platelet activation, and a sequential inflammatory cascade [1-3]. Notably, polymorphonuclear neutrophils (PMNs) disrupt and inhibit the activity of fibrin, fibrinogen, platelets, and other coagulation factors, which leads to increased blood loss and transfusion requirements [4]. Ulinastatin (Ulistin; HanLim Pharmaceutical Co., Seoul, Korea) is a nonspecific protease inhibitor, also a urinary trypsin inhibitor, and a type of glycoprotein that is extracted and purified from fresh human urine [5]. It represses inflammatory activity, permeation of neutrophils, and release of elastase and chemical mediators [6]. One study reported that ulinastatin normalizes the coagulation function and prevents changes in thromboelastography (TEG) during liver resection surgery [7]. Furthermore, ulinastatin may shorten prothrombin time (PT), activated partial thromboplastin time (aPTT), and activated coagulation time in patients undergoing CPB [8]. The present study aimed to evaluate whether the intraoperative administration of ulinastatin could improve the early coagulation profile, postoperative blood loss, or transfusion requirements of patients undergoing atrioventricular valve surgery using CPB and assess the effect of ulinastatin on major organ dysfunction, systemic inflammatory activities, and other postoperative profiles. BODY.MATERIALS AND METHODS: BODY.1) PATIENTS: All patients undergoing cardiac surgery are prospectively registered at Konkuk University Medical Center. These registries prospectively contain baseline characteristics of patients, perioperative evaluation data, and the results and any complications of surgery. This study was approved by Konkuk University Medical Center independent institutional review board. A total of 425 patients underwent cardiac surgery from January 2008 through February 2009. We excluded patients who had re-do cardiac surgery, severe hepatic or pulmonary disease, left ventricular ejection fraction <40%, pre-existing renal dysfunction (serum creatinine level >2.0 mg/dL), those older than 80 years of age, and those who had been treated with antithrombotic agents within 2 weeks of surgery. Among them, 110 patients who had atrioventricular valve surgery through right thoracotomy were enrolled in the present study. These patients were assigned to either the ulinastatin group (n=41) or the control group (n=69), and this review was done retrospectively (Table 1). BODY.2) CARDIOPULMONARY BYPASS: All of the patients received standardized CPB management in the same manner. Systemic heparinization (300 U/kg intravenously) and arterial and venous cannulations were performed at an activated clotting time (ACT) >450 seconds, which was measured by the Hemochron (International Technidyne Co., Edison, NJ, USA). CPB was initiated with a membrane oxygenator primed with normal saline with 20% mannitol, 6 mEq sodium bicarbonate, 20% albumin, 5,000 U heparin, 1.5 g cefuroxime, and 2 g calcium gluconate (total priming volume, 20 mL/kg). Steroids were not applied during operation and the ACT was maintained at >450 seconds during the CPB procedure. The CPB flow was initiated at a rate of 60 mL/kg/min and was adjusted according to the state of hemodilution and core temperature. Myocardial protection was achieved by means of antegrade cold blood cardioplegic solution (20 mL/kg). The blood cardioplegic solution included sodium chloride (6.43 g/L), potassium chloride (1.193 g/L), calcium chloride (0.176 g/L), and magnesium chloride (3.253 g/L) at conditions of pH 7.4 and 4℃ to 8℃. Moderate systemic hypothermia (range, 28℃ to 30℃) was used. Patients were weaned from CPB when the rectal temperature reached 35℃. Protamine (3 mg/kg) was applied intravenously with reversal of heparinization after CPB. BODY.3) SURGICAL PROCEDURE: Right thoracotomy was performed on patients of atrioventricular valve diseases. In thoracotomy, we mostly used the femoral artery and vein or right internal jugular vein as vascular access for CPB. More details about the thoracotomy approach procedure have been described in a previous paper [9]. The amounts of chest tube drainage were recorded every hour for 6 hours after admittance to the intensive care unit (ICU). If the accumulated amounts were over 400 mL within the first hour postoperatively, at a rate of over 5 mL/kg/hr for 3 consecutive hours, or >10 mL/kg/hr at any time, it warranted surgical re-exploration for bleeding control. BODY.4) ULINASTATIN ADMINISTRATION: Randomly selected patients of the ulinastatin group were treated with 5,000 U/kg ulinastatin (Ulistin) just prior to ACC. In contrast, patients in the control group received an equivalent volume of normal saline as a placebo. BODY.5) TRANSFUSION: The threshold for transfusion of packed red blood cells was a hematocrit <20% during CPB and <30% after CPB. The collected blood derived from the CPB circuit was salvaged by a cell salvage device after reversal of heparinization. Fresh frozen plasma (FFP) was transfused when the postoperative international normalized ratio (INR) was 1.5 to 2.0 (1 unit FFP) or >2.0 (2 units FFP). Platelets (8 units) were transfused when the postoperative platelet count was <50,000/µL. Meanwhile, cryoprecipitate (10 units) was transfused when the postoperative fibrinogen count was <100 mg/dL. The total transfusion quantities were recorded for 24 hours after arrival at the ICU after the operation. TEG (Hemonetic Co., Niles, IL, USA) was routinely used in cardiac surgery as viscoelastic hemostatic assays (VHA). Many studies have demonstrated the superiority of using VHA as compared to routine coagulation tests both in predicting bleeding and the need for re-do surgery and in reducing the total amount of blood transfusions [10]. In this trial, the TEG study was performed just before the initiation of anesthesia and on arrival at the ICU after operation. BODY.6) INTENSIVE CARE UNIT MANAGEMENT: All of the patients were transferred to the ICU after surgery, where they received appropriate management. Fluid therapy was administered to maintain the pulmonary capillary wedge pressure at 9 to 15 mmHg, the cardiac index >2.0 L/min/m2, and the urine output >0.5 mL/kg/hr. The duration of ventilator support, length of ICU stay, entire hospitalization period, and other hemodynamic variables were recorded. The patients were weaned off the ventilator when they met the following criteria: hemodynamic stability; urine output ≥0.5 mL/kg/hr; chest tube drainage <100 mL/hr; oxygen saturation (SpO2) at pulse oximetry of ≥95% at a fraction of inspired oxygen (FiO2) of ≤0.5; pH ≥7.3 and arterial carbon dioxide ≤55 mmHg from arterial blood gas analysis; no neurologic abnormal signs; and appropriate response to commands. BODY.7) STATISTICAL ANALYSES: Statistical analyses were performed with SPSS ver. 18.0 (SPSS Inc., Chicago, IL, USA). Categorical variables are expressed as percentages or numbers, and continuous variables are expressed as means with standard deviations. After testing for normality of distribution, continuous variables were compared using the Student t-test. Categorical variables were compared using Fisher's exact test. The Wilcoxon signed-rank test was used to compare values before and after surgery within the same group. All p-values less than 0.05 were considered statistically significant. BODY.RESULTS: Patients in both groups had similar preoperative characteristics. CPB time, ACC time, total dosage of heparin, and protamine administration were not significantly different between the two groups (Table 2). The postoperative platelet count was statistically lower in the ulinastatin group compared with the control group; however, there were no significant differences in PT, INR, aPTT, or TEG. Cardiac markers (isoenzyme creatine kinase with muscle and brain subunits [CK-MB] or troponin-I) and the brain natriuretic peptide level were significantly higher in both groups than the respective preoperative values (p<0.05), but there were no significant differences between the two groups. There were no statistical differences between fibrinogen, creatinine, lactate, and aspartate aminotransferase (AST) in the two groups (Table 3). Among the postoperative outcomes, no statistical significant differences were observed between the two groups in the duration of mechanical ventilator support, ICU stay, chest tube drainage for postoperative 6 hours, chest tube indwelling time, hospital stay, transfusion amounts, or postoperative complications (p>0.05) (Table 4). BODY.DISCUSSION: Urinary trypsin inhibitor, which is also called ulinastatin, is a sort of glycoprotein with a molecular weight of about 24,000 Da, and is extracted and purified from fresh human urine [11]. It consists of 143 amino acid residues and two Kunitz-type protease-inhibitor domains arranged in tandem and represents the light chain of inter-α-trypsin inhibitor existing in blood. Ulinastatin has anti-inflammatory activity, and suppresses the infiltration of neutrophils and the release of inflammatory chemical mediators such as polymorphonuclear neutrophil elastase (PMNE), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-8 [12]. In this study we did not observe any beneficial effects of ulinastatin on early coagulation profiles or postoperative blood loss. Furthermore, there were no significant differences in the postoperative laboratory and clinical outcomes between the control and ulinastatin groups. Ulinastatin is presumed to reduce postoperative blood loss and transfusion requirements because it is a kind of protease inhibitor that is similar to aprotinin. Previously, a few studies have documented that ulinastatin reduced postoperative blood loss in major orthopedic surgery and showed a non-significant trend towards decreased blood loss in gastrectomy [13]. The extent of the systemic inflammatory reaction actually varies according to the type of surgery and the use of CPB. Therefore, the present outcomes in this study may differ from those published previously. Unlike previous reports related to orthopedic or abdominal surgery, this study focuses on patients who underwent open heart surgery with CPB. CPB activates more extensive systemic inflammatory responses than other surgeries and may overmaster the anti-inflammatory capacity of ulinastatin [3,14]. Ulinastatin is also known to prevent increases in CK-MB, creatinine, and lactate after severe burn injury [15]. Several other studies have suggested that ulinastatin can reduce ischemic and reperfusion injuries of major organs such as the kidneys, lungs, and liver [16,17]. Unlike animal experiments (15,000 U/kg) and pediatric studies (12,000 U/kg), however, adult cardiac surgery studies (range, 5,000 to 6,000 U/kg) have reported conflicting results. According to these studies, doses of ulinastatin administered to adult patients were relatively small compared to the others, and assumed to be insufficient to provide a complete anti-inflammatory effect. Likewise, the present study (5,000 U/kg) revealed no significant effect of ulinastatin on cardiac markers, creatinine, or lactate levels, though postoperative levels of cardiac markers and lactate were significantly higher than preoperative ones. These results strongly suggest that we need to consider the increased dose of ulinastatin concentrations to estimate the effect of prevention from major organ failure. Another previous study reported that ulinastatin attenuated the elevation of IL-8 release after CPB and was closely associated with improved pulmonary variables. Ulinastatin also prevented increases in the alveolar-arterial oxygen difference after CPB and preserved pulmonary function by inhibiting the release of elastase, TNF-α, IL-6, and IL-8 [18]. In this study, cardiac operations performed via right thoracotomy were screened out in order to judge how much the administration of ulinastatin aids in postoperative respiratory recovery. Nevertheless, there were no relevant records of pulmonary parameters such as the respiratory index, intrapulmonary shunt (QST/QT), and PaO2/FIO2 fraction ratio, so we analyzed hemodynamic variables, the total duration of mechanical ventilator support, and ICU stay [19]. Another study reported that the duration of ICU stay was significantly shorter in patients treated with ulinastatin than in controls because of improvement in pulmonary function [20]. However, no significant differences were observed between the two groups in the present review. As mentioned above, it is well known that ulinastatin inhibits the activity of PMNs, but this study did not measure parameters that reflect the activity of PMNs, such as elastase. One of the critical limitations of this study was that we could not assess whether the administered dose of ulinastatin suppressed the release of PMNE adequately. Overall, the present study showed that intra-operative application of ulinastatin (5,000 U/kg) did not influence systemic inflammatory and coagulative reactions or multi-organ dysfunctions, especially postoperative blood loss and transfusion requirements. As far as the present study, an insufficient dosage of administered ulinastatin could be considered first, and a continuous infusion of high-dose ulinastatin could be more effective because the plasma half-life of ulinastatin is short, at about 40 minutes [21]. A further extensive study and examination of the dosage and interval time of ulinastatin is required to warrant the validity of appropriate administration. BODY.CONCLUSION: In conclusion, the present study revealed no significant effect of ulinastatin on major organ dysfunction, systemic inflammatory reactions, and other postoperative profiles. Additionally, ulinastatin did not improve the early coagulation profile, postoperative blood loss, or transfusion requirements of patients undergoing open heart surgery with CPB and ACC.

TITLE: Immunogenicity and Safety of a Quadrivalent Meningococcal Serogroups A, C, W-135 and Y Tetanus Toxoid Conjugate Vaccine (MenACWY-TT) Administered to Adults Aged 56 Years and Older: Results of an Open-Label, Randomized, Controlled Trial ABSTRACT.BACKGROUND: The burden of invasive meningococcal disease is substantial in older adults in whom the case fatality rate is high. Travelers to regions with high rates of meningococcal disease, such as Hajj pilgrims, are at increased risk of meningococcal infection, and disease transmission from travelers to their close contacts has been documented. In younger individuals, meningococcal conjugate vaccines offer advantages over polysaccharide vaccines in terms of duration of protection and boostability, and induction of herd immune effects through reductions in nasopharyngeal carriage of meningococci. To date, few data are available evaluating meningococcal conjugate vaccine use in adults >55 years of age. ABSTRACT.OBJECTIVE: To evaluate the immunogenicity and safety of quadrivalent meningococcal serogroups A, C, W-135 and Y vaccine with all serogroups conjugated to tetanus toxoid (MenACWY-TT, Nimenrix™, GlaxoSmithKline, Belgium) and a licensed quadrivalent polysaccharide vaccine (MenPS, Mencevax™ GlaxoSmithKline, Belgium) in adults >55 years of age. ABSTRACT.METHODS: This was a phase IIIb, open-label, randomized (3:1), controlled study conducted at one study center in Lebanon. A total of 400 healthy adults between 56 and 103 years of age without previous MenPS or tetanus toxoid vaccination within the previous 5 years or meningococcal conjugate vaccination at any time previously were included. They received a single-dose vaccination with MenACWY-TT or MenPS with blood sampling before and 1 month after vaccination. The main outcome measures were serum bactericidal activity (rabbit complement source: rSBA) vaccine response (VR) rate [rSBA titer of ≥1:32 in initially seronegative subjects (rSBA titer <1:8); ≥4-fold increase in subjects with pre-vaccination rSBA titers between 1:8 and 1:128, and ≥2-fold increase in subjects with pre-vaccination rSBA titers ≥1:128]. The percentages of subjects with rSBA titers ≥1:8 and ≥1:128 and rSBA geometric mean titers (GMTs) were assessed. Solicited adverse events were recorded for 4 days following vaccination, and all other adverse events, including the incidence of new onset chronic diseases, were recorded for 31 days after vaccination. ABSTRACT.RESULTS: One month after a single dose of MenACWY-TT, the rSBA VR rate in the MenACWY-TT group was 76.6 % for serogroup A, 80.3 % for serogroup C, 77.5 % for serogroup W-135 and 81.9 % for serogroup Y. VR rates in the MenPS group were 91.7, 84.8, 87.1 and 89.1 %, respectively. One month after vaccination, ≥93.2 % of subjects in the MenACWY-TT group and ≥93.9 % in the MenPS group had rSBA titers ≥1:128. In each group, GMTs increased by ≥13-fold for each serogroup. rSBA VR and GMTs tended to be lower in subjects who were over 65 years compared to 56–65 years of age. Only 6.3 % of MenACWY-TT recipients had anti-TT ≥0.1 IU/ml prior to vaccination, increasing to 28.1 % post-vaccination. The rSBA GMTs were 1.9- to 4-fold higher in anti-TT responders. Each local and general solicited symptom was reported by no more than 3.0 % of subjects in either group. No serious adverse events were considered vaccine related. ABSTRACT.CONCLUSION: In adults 56 years of age and older, MenACWY-TT was immunogenic, with a vaccine response rate ≥76 % and with ≥93 % of subjects achieving rSBA titers ≥1:128 against all four serogroups after a single dose. MenACWY-TT induced low anti-TT concentrations in this population, which deserves further study. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s40266-013-0065-0) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Invasive disease caused by Neisseria meningitidis is typically characterized by rapid onset with fulminant progression, resulting in death in around 10 % of individuals despite appropriate antibiotics and supportive care [1]. The incidence of invasive meningococcal disease is highest in infants, with a second peak that occurs during adolescence [2]. Notwithstanding, a substantial proportion of the disease burden lies with adults: in the US approximately 43 % of all invasive meningococcal disease cases were reported in adults aged 25 years and older (1998–2007 data) [2]. During the same observation period, 14 % of cases occurred in the age group 65 years and older, with a case fatality rate of 23.8 %, the highest in any age group [2]. Similar trends are observed in other countries: in Australia 6 % of all meningococcal cases in 2010 were in adults over 65 years of age, and 19 % were in adults 45 years of age and older [3]. In England and Wales, around 10 % of all meningococcal cases each year between 1993 and 2004 occurred in adults over 45 years of age [4]. These data point to a substantial disease burden in older adults, which is likely to become a greater public health concern as the global population ages. Based on differences in the composition of the polysaccharide capsule, 12 meningococcal serogroups have been identified; however, 6 serogroups predominate as causes of invasive disease (serogroups A, B, C, W-135, X and Y) [5]. In the Middle East, available information suggests that serogroups A and W-135 are responsible for the majority of meningococcal invasive disease in the region. Serogroups A and W-135 are also responsible for major epidemics in Africa [1, 5], while serogroup X emerged more recently as a cause of local outbreaks in Africa [6–9]. By contrast, serogroups A, W-135 and X are infrequently identified in many Western countries, where serogroups B, C and to a lesser degree Y predominate [2, 5]. Adults are at increased risk of meningococcal infection when they travel to meningococcal endemic regions [10]. A serogroup W-135 outbreak that occurred during the 2000 Hajj spread intercontinentally from pilgrims to their contacts [11, 12], and more recently, cases of W-135 were identified in France among travellers who had returned from West Africa and their contacts [13]. These cases of intercontinental transmission highlight the relative ease by which meningococcal strains can be transmitted to immunologically naïve populations through global travel activity. Meningococcal vaccination against serogroups A, C, W-135 and Y is now required prior to Hajj attendance for all pilgrims over 2 years of age [14]. Vaccination is the best strategy to prevent meningococcal disease. Quadrivalent serogroups A, C, W-135 and Y (ACWY) meningococcal polysaccharide vaccines are available for use in adults and children from the age of 2 years [15]. However, polysaccharide vaccines do not stimulate T-cell-dependent immune responses and thus do not induce long-lasting protection or immune memory, but may induce immune hyporesponsiveness, particularly for serogroup C, on repeated administration [16, 17]. By contrast, meningococcal conjugate vaccines induce T-cell-mediated immune responses that appear to be long-lived, with evidence of immune memory upon re-exposure [15]. Long-term protection against invasive meningococcal disease appears to be dependent on the presence of circulating antibody rather than on memory responses [18]. Antibody levels decrease over time after both meningococcal polysaccharide and meningococcal conjugate vaccination, and the duration of protection afforded by meningococcal conjugate vaccination is not yet known. Conjugate vaccines also induce herd protection by decreasing the carriage rate in vaccine recipients, which was demonstrated in the UK after a mass vaccination program with conjugate serogroup C vaccines [19]. Herd protection is important in interrupting the transmission of meningococci. Meningococcal conjugate vaccines therefore have advantages in terms of duration of protection and boostability in those who require sustained protection. In adults, meningococcal ACWY (MenACWY) conjugate vaccines show comparable or improved immunogenicity versus polysaccharide vaccines [20–22]. However, few studies have evaluated conjugate vaccine use in adults over 55 years of age [20]. GlaxoSmithKline has developed a MenACWY vaccine with all serogroups conjugated to tetanus toxoid (MenACWY-TT, Nimenrix™), which is licensed for use in Europe as a single dose in individuals as of 1 year of age [23]. MenACWY-TT was immunogenic and well tolerated in clinical trials conducted in children, adolescents and adults up to age 55 years [21, 22, 24–30]. We conducted a phase IIIb, open-label, randomized, controlled study designed to evaluate the immunogenicity and safety of MenACWY-TT as compared to a licensed polysaccharide vaccine (Mencevax™ACWY, GlaxoSmithKline Vaccines: MenPS) when administered as one dose to healthy adults aged 56 years and older. BODY.METHODS: BODY.STUDY DESIGN: The study (113807, www.clinicaltrials.gov NCT01235975) was conducted in a single center in Lebanon between 30 November 2010 and 3 August 2011. Adults were enrolled and randomized 3:1 into two parallel groups. Subjects in the MenACWY-TT group received a single dose of MenACWY-TT, while subjects in the MenPS group received a single dose of licensed MenPS. A randomization list was generated at GlaxoSmithKline Belgium and was used to number the vaccines. Treatment allocation at the investigator site was performed using a central, web-based randomization system. A blocking scheme ensured that balance between treatments was maintained. The randomization algorithm used a minimization procedure accounting for center and age strata. The enrollment ensured a 2:1 allocation of the population across two age strata: 56–65 years of age and >65 years of age. This was an open study because the routes of administration of the study vaccine and the control vaccine were different. The primary study objective was to evaluate the immunogenicity of MenACWY-TT as compared to MenPS in terms of serum bactericidal activity (rabbit complement source; rSBA) vaccine response (VR) rates 1 month after vaccination. A VR was defined as an rSBA titer of at least 1:32 in initially seronegative subjects (rSBA titer <1:8), at least a four-fold increase in subjects with rSBA titers between 1:8 and 1:128 before vaccination and at least a two-fold increase in subjects with rSBA titers ≥1:128 before vaccination. BODY.STUDY SUBJECTS: Adults were to be at least 56 years of age at enrollment, and females were to be of non-child bearing potential. Subjects were not enrolled if they had received prior vaccination with MenPS or tetanus toxoid (TT) within the previous 5 years or with any meningococcal conjugate vaccine at any time previously. Subjects were also excluded if they had previously suffered from meningococcal disease, if they were immunosuppressed for any reason including chronic (>14 days) immunosuppressant treatment, if they had a history of neurological disease, seizures or Guillain-Barre syndrome, or if they had active pulmonary, cardiovascular, hepatic or renal disease. Subjects who had received blood products within 3 months of vaccination or who had chronic alcohol consumption or drug abuse were also ineligible to participate. BODY.STUDY VACCINES: One 0.5 ml dose of MenACWY-TT contained 5 μg each of meningococcal serogroup A, C, W-135 and Y polysaccharide conjugated to a total of approximately 44 μg TT. One 0.5-ml dose of MenPS contained 50 μg each of meningococcal serogroups A, C, W-135 and Y polysaccharide. MenACWY-TT was administered intramuscularly and MenPS given subcutaneously into the upper arm on the non-dominant side. BODY.IMMUNOGENICITY ASSESSMENT: Blood samples were collected from all subjects before and 1 month after vaccination and were tested for rSBA for each meningococcal serogroup as previously described [31]. The cutoff of the rSBA assay was a 1:8 dilution and was considered indicative of seroprotection [32, 33]. Anti-TT antibodies were measured at each time point using an enzyme-linked immunosorbent assay (ELISA) [34], with an assay cutoff of 0.1 IU/ml. BODY.SAFETY AND REACTOGENICITY ASSESSMENT: Specific local and general symptoms were recorded on diary cards for 4 days after vaccination. Symptom intensity of redness, swelling and fever was graded by millimeter of reaction and degrees Celsius of fever, respectively, and all other symptoms were graded by the subject using a pre-defined scale. Grade 3 redness and swelling were defined as diameter >50 mm, grade 3 fever as oral temperature >39.5°C, and for other symptoms, grade 3 was defined as preventing normal activity. All other adverse events including serious adverse events and new onset of chronic illness were recorded for 31 days after vaccination. BODY.CONSENT PROCEDURES: The study protocol was approved by the institutional review board (IRB) prior to study start. Written informed consent was obtained from each subject prior to enrollment. Enrollment was hampered by the distance between home communities and the study center in Beirut. In response, the principal investigator proposed setting up a mobile unit to perform study visits by study personnel in local villages, as per protocol. A mobile unit guidance explaining the rationale for the mobile unit and the associated procedures was issued to the IRB for their information and the enrollment via the mobile unit started on the same day. Later, the IRB clarified that approval of the mobile unit was required. The IRB approved the mobile unit pending minor revisions a few days later. One hundred and two subjects were enrolled through the mobile unit prior to formal IRB mobile unit approval. All of these subjects were asked to re-consent. Twenty-nine subjects didn’t re-consent (no subjects refused to re-consent because of an adverse event), and therefore these 29 subjects were eliminated from all statistical analyses. BODY.STATISTICAL ANALYSES: As this is the first study to investigate the immunogenicity of MenACWY-TT in a population of older adults, this exploratory study was designed as a descriptive analysis. The primary analysis of immunogenicity was done on the according-to-protocol (ATP) cohort for immunogenicity, which included subjects who had complied with all protocol-defined procedures and who had data available for at least one immunogenicity endpoint. Percentages of subjects with rSBA vaccine response and with titers/concentrations above threshold values, with exact 95 % CIs, were calculated. The effect of age at vaccination and of meningococcal vaccination history was investigated in exploratory analyses. The target enrollment was set at 400 subjects randomized 3:1 to the MenACWY-TT group or MenPS group in order to have 360 evaluable subjects (270 subjects in MenACWY-TT group and 90 subjects in MenPS group), assuming a 10 % drop out of subjects throughout the study. With 270 evaluable subjects in the MenACWY-TT group and assuming a VR rate of 80 %, the exact 95 % CI around the estimate would be 74.7; 84.6. With 90 evaluable subjects in the MenPS group, the 95 % CI around the assumed VR rate of 80 % would be 70.2; 87.7. Exploratory analyses were used to highlight potential differences between groups if the asymptotic standardized 95 % CI for the group difference in the percentage of subjects reaching specified immunological cutoffs did not contain the value ‘0’ or if the 95 % CI for the geometric mean titer (GMT) ratio (ANCOVA model using the pre-vaccination logarithm10 transformation of the titers/concentrations, the vaccine group, age strata and meningococcal vaccination history as covariates) between groups did not contain the value ‘1’. Multiple comparisons were done without adjustment for multiplicity, and there is a risk that statistically significant differences may have occurred by chance alone. Thus, any statistically significant differences between groups should be interpreted cautiously. Exploratory analyses for statistical differences were performed on the cohort including all subjects; these analyses were not performed per age strata because of the limited available sample size per stratum. The primary analysis of safety was done on the total vaccinated cohort, comprising all vaccinated subjects with safety data available. Statistical analyses were performed using SAS® software version 9.2 (SAS Institute Inc., Cary, NC, USA) and ProcStatXact 8.1. BODY.RESULTS: BODY.STUDY SUBJECTS: Four hundred adults whose age ranged between 56 and 103 years were enrolled in the study. Of these, 2 subjects were not vaccinated, and 29 were excluded from all statistical analyses (see Sect. 2). Thus, there were 369 subjects in the total vaccinated cohort, of which 260 (70 %) were eligible for inclusion in ATP analyses (Fig. 1). More females (68.3 %) than males (31.7 %) were enrolled. The two treatment groups were comparable in terms of demographic characteristics (Table 1).Fig. 1Subject flow through the study. ATP according to protocol, SAE serious adverse event. *Twenty-nine subjects (randomized and vaccinated) who were enrolled via the mobile unit prior to approval and who did not re-consent after mobile unit approval by the institutional review board Table 1Summary of demographic characteristics (total vaccinated cohort) CharacteristicMenACWY-TT N = 274MenPS N = 95Age (years)  Mean (SD) 64.1 (7.22) 64.3 (7.39)  Range 56–103 56–86 Age stratum (years) n (%)  56–65 184 (67.2) 63 (66.3)  >65 90 (32.8) 32 (33.7) Gender n (%)  Female 182 (66.4) 70 (73.7)  Male 92 (33.6) 25 (26.3) Geographic ancestry n (%)  Arabic/North African 274 (100) 95 (100) Meningococcal vaccination historyan (%)  Unknown 2 (0.7) 2 (2.1)  0 210 (76.6) 68 (71.6)  1 62 (22.6) 25 (26.3)  2 0 (0.0) 0 (0.0) N total number of subjects, n/% number/percentage of subjects in a given category, SD standard deviation aNumber of previous meningococcal polysaccharide vaccinations. Note that previous meningococcal vaccines were received more than 5 years prior to enrollment One subject (an 84-year-old subject in the MenPS group) withdrew from the study because of a serious adverse event. This subject suffered a cerebrovascular accident and died 21 days after vaccination. The event was considered unrelated to vaccination by the investigator. BODY.IMMUNOGENICITY: After a single dose of MenACWY-TT, the VR rate in the MenACWY-TT group was 76.6 % for serogroup A, 80.3 % for serogroup C, 77.5 % for serogroup W-135 and 81.9 % for serogroup Y (Table 2). VR rates in the MenPS group were 91.7, 84.8, 87.1 and 89.1 %, respectively.Table 2Percentage of subjects with a vaccine response 1 month after vaccination: all subjects and by age strata (ATP cohort for immunogenicity) SerogroupGroupAll subjects56- to 65-year-olds>65-year-oldsNn% [95 % CI]Nn% [95 % CI]Nn% [95 % CI]A MenACWY-TT 175 134 76.6* [69.6; 82.6] 119 93 78.2 [69.6; 85.2] 56 41 73.2 [59.7; 84.2] MenPS 60 55 91.7 [81.6; 97.2] 37 37 100 [90.5; 100] 23 18 78.3 [56.3; 92.5] C MenACWY-TT 188 151 80.3 [73.9; 85.7] 128 106 82.8 [75.1; 88.9] 60 45 75.0 [62.1; 85.3] MenPS 66 56 84.8 [73.9; 92.5] 41 38 92.7 [80.1; 98.5] 25 18 72.0 [50.6; 87.9] W-135 MenACWY-TT 187 145 77.5 [70.9; 83.3] 127 102 80.3 [72.3; 86.8] 60 43 71.7 [58.6; 82.5] MenPS 62 54 87.1 [76.1; 94.3] 38 33 86.8 [71.9; 95.6] 24 21 87.5 [67.6; 97.3] Y MenACWY-TT 188 154 81.9 [75.7; 87.1] 128 103 80.5 [72.5; 86.9] 60 51 85.0 [73.4; 92.9] 64 57 89.1 [78.8; 95.5] 39 37 94.9 [82.7; 99.4] 25 20 80.0 [59.3; 93.2] Vaccine response defined as: post-vaccination rSBA titer ≥1:32 in initially seronegative subjects; four-fold increase in initially seropositive subjects with rSBA titer 1:8 and <1:128; two-fold increase in initially seropositive subjects with rSBA titer ≥1:128 ATP according-to-protocol, N number of subjects with both pre- and post-vaccination results available, n/% number/percentage of responders; 95 % CI 95 % confidence interval, rSBA rabbit complement source * Statistically significant difference between the MenACWY-TT and MenPS groups (exploratory analysis) One month after vaccination, at least 97.4 % in the MenACWY-TT group and at least 95.5 % in the MenPS group had rSBA titers ≥1:8 (Table 3). The percentage with rSBA ≥1:128 was at least 93.2 % in the MenACWY-TT group and at least 93.9 % in the MenPS group. In each group, GMTs increased by at least 13-fold for each serogroup (Table 3).Table 3Percentage of subjects with rSBA titers ≥1:8 and ≥1:128 and GMTs (ATP cohort for immunogenicity) SerogroupGroupTime pointN≥1:8≥1:128GMT [95 % CI]n% [95 % CI]n% [95 % CI]A MenACWY-TT Pre 181 136 75.1 [68.2; 81.3] 104 57.5 [49.9; 64.8] 108.3 [77.9; 150.5] Post 186 185 99.5 [97.0;100] 177 95.2 [91.0; 97.8] 1,442.3* [1,174.4; 1,771.3] MenPS Pre 61 43 70.5 [57.4; 81.5] 37 60.7 [47.3; 72.9] 102.1 [55.2; 188.7] Post 65 65 100 [94.5; 100] 63 96.9 [89.3; 99.6] 2,840.1 [2,062.3; 3,911.1] C MenACWY-TT Pre 190 135 71.1 [64.0; 77.4] 86 45.3 [38.0; 52.6] 71.5 [51.1; 100.1] Post 192 192 100 [98.1; 100] 179 93.2 [88.7; 96.3] 2,498.6* [1,887.0; 3,308.2] MenPS Pre 66 44 66.7 [54.0; 77.8] 29 43.9 [31.7; 56.7] 73.8 [38.0; 143.6] Post 66 65 98.5 [91.8; 100] 62 93.9 [85.2; 98.3] 4,815.1 [2,827.0; 8,201.2] W-135 MenACWY-TT Pre 188 135 71.8 [64.8; 78.1] 98 52.1 [44.7; 59.5] 84.7 [61.1; 117.3] Post 193 188 97.4 [94.1; 99.2] 183 94.8 [90.7; 97.5] 1,454.0 [1,130.5; 1,870.1] MenPS Pre 62 40 64.5 [51.3; 76.3] 30 48.4 [35.5; 61.4] 68.5 [37.4; 125.3] Post 66 63 95.5 [87.3; 99.1] 62 93.9 [85.2; 98.3] 1,838.4 [1,134.6; 2,978.9] Y MenACWY-TT Pre 189 148 78.3 [71.7; 84.0] 118 62.4 [55.1; 69.4] 137.6 [100.7; 187.9] Post 193 193 100 [98.1; 100] 187 96.9 [93.4; 98.9] 2,547.0 [2,059.6; 3,149.8] MenPS Pre 64 55 85.9 [75.0; 93.4] 46 71.9 [59.2; 82.4] 217.4 [131.7; 358.9] Post 66 66 100 [94.6; 100] 65 98.5 [91.8; 100] 3,931.6 [2,726.1; 5,670.2] ATP according-to-protocol, GMT geometric mean antibody titer calculated on all subjects, N number of subjects with available results, n/% number/percentage of subjects with titer within the specified range, 95 % CI 95 % confidence interval, Pre Pre-vaccination, Post 1 month post-vaccination, rSBA rabbit complement source * Statistically significant difference between the MenACWY-TT and MenPS groups (exploratory analysis) Exploratory analyses did not detect any statistically significant differences between groups in terms of the percentage of subjects who reached the 1:8 and 1:128 thresholds after vaccination. However, these analyses suggested that the magnitude of the response was statistically significantly lower in MenACWY-TT recipients than in MenPS recipients in terms of VR rate for serogroup A and rSBA GMTs for serogroups A and C. VR rates in individuals with pre-existing rSBA titers ≥1:128 tended to be lower for each serogroup in MenACWY-TT recipients (65.7–72.6 %) than in MenPS recipients (75.9–91.3 %), while the majority of initially seronegative subjects in both groups demonstrated VRs to each serogroup after vaccination (for serogroup A, 93.3 % in the MenACWY-TT group and 100 % in the MenPS group; for serogroup C, 96.3 and 90.9 %; for W-135, 88.7 and 86.4 %; and for Y, 100 % in both groups). BODY.EFFECT OF AGE AND PREVIOUS MENINGOCOCCAL POLYSACCHARIDE VACCINATION ON THE IMMUNE RESPONSE: VR rates tended to be lower in subjects who were >65 years of age at the time of vaccination, compared to younger (56- to 65-year-old) subjects (Table 2). This trend was more pronounced in MenPS recipients for serogroups A, C and Y. Possible differences between groups were not statistically tested as these tests are not powered to do so, because of the low numbers of subjects in each subgroup. The percentage of subjects with rSBA titers ≥1:128 and the rSBA GMT were within the same range in the 56–65 year and >65 year subgroups, although the small number of subjects in each group means that conclusions cannot be drawn (Supplementary table 1). Approximately one-quarter of subjects had a history of having received a meningococcal polysaccharide vaccine more than 5 years previously (Table 1). Prior to vaccination, the percentage of subjects with rSBA ≥1:128 was between 57.5 and 60.7 % for serogroup A, 43.9 and 45.3 % for serogroup C, 48.4 and 52.1 % for serogroup W-135 and 62.4 and 71.9 % for serogroup Y (Table 3). The percentage of subjects with pre-vaccination titers ≥1:128 and rSBA GMTs for serogroups A and C, but not W-135 or Y, tended to be higher in subjects with a history of meningococcal vaccination than in unvaccinated subjects (Supplementary table 2). In the MenACWY-TT group, the post-vaccination rSBA GMTs in subjects who had not received a meningococcal dose previously were in the same range as in subjects who had received a prior meningococcal vaccination, although lower point values for rSBA-W-135 titers were observed in those previously vaccinated: for serogroup A, 1,372.0 [95 % CI 1,088.3; 1,729.6] versus 1,726.4 [1,067.5; 2,792.0]; for serogroup C, 2,584.3 [1,836.2; 3,637.2] versus 2,412.1 [1,504.7; 3,866.7]; for serogroup W-135, 1,791.7 [1,369.7; 2,343.7] versus 756.2 [406.2; 1,407.7]; and for serogroup Y, 2,664.0 [2,107.0; 3,368.3] versus 2,219.6 [1,324.1; 3,720.6]. In the MenPS group, the rSBA GMTs were: for serogroup A, 2,640.3 [1,768.5; 3,941.9] versus 3,968.1 [2,248.5; 7,003.0]; for serogroup C, 5,054.1 [2,661.9; 9,595.9] versus 4,712.0 [1,483.6; 14,965.4]; for serogroup W-135, 2,217.4 [1,363.6; 3,606.0] versus 1,325.9 [334.1; 5,261.7]; and for serogroup Y, 4,370.4 [2,756.7; 6,928.6] versus 3,710.3 [2,042.1; 6,741.0]. Due to the low numbers of subjects in each subgroup, comparisons between groups are not reliable (Supplementary table 2). BODY.RESPONSE TO TETANUS TOXOID: Few subjects had anti-TT antibodies ≥0.1 IU/ml prior to vaccination (12 subjects, 6.3 % in the MenACWY-TT group and 6 subjects, 9.1 % in the MenPS group). The anti-TT response after MenACWY-TT vaccination was low (Table 4).Table 4Percentage of subjects with anti-TT concentrations ≥0.1 and 1 IU/ml and GMCs (ATP cohort or immunogenicity) GroupTimingN≥0.1 lU/ml≥1 lU/mlGMC [95 % CI]n% [95 % CI]n% [95 % CI]MenACWY-TT Pre 192 12 6.3 [3.3; 10.7] 3 1.6 [0.3; 4.5] 0.058 [0.053; 0.063] Post 192 54 28.1 [21.9; 35.1] 29 15.1 [10.4; 21.0] 0.137 [0.104; 0.180] MenPS Pre 66 6 9.1 [3.4; 18.7] 1 1.5 [0.0; 8.2] 0.060 [0.051; 0.070] Post 66 6 9.1 [3.4; 18.7] 1 1.5 [0.0; 8.2] 0.060 [0.051; 0.071] ATP according-to-protocol, GMC geometric mean antibody concentration calculated on all subjects, N number of subjects with available results, n/% number/percentage of subjects with concentration within the specified range, 95 % CI 95 % confidence interval, Pre Pre-vaccination, Post 1 month post-vaccination, TT tetanus toxoid A post-hoc analysis assessed rSBA GMTs in subjects according to pre- and post-vaccination anti-TT antibody status in the MenACWY-TT group. In adults with anti-TT antibodies ≥0.1 IU/ml prior to vaccine, post-vaccination rSBA GMTs after MenACWY-TT were as high or higher than those observed in the MenPS group for serogroups C, W-135 and Y, but not for serogroup A (rSBA GMT 2,479.8 [95 % CI 1,211.4; 5,076.2] for serogroup A, 7,492.7 [2,069.9; 27,121.6] for serogroup C, 3,250.5 [521.4; 20,263.8] for serogroup W-135, and 3,992.0 [1,028.7; 15,491.2] for serogroup Y, Supplementary table 3). Furthermore, the post-vaccination rSBA GMTs were between 1.9- and 4-fold higher in subjects who responded to TT: That is, the point values of the rSBA GMT for each serogroup was higher (no statistical test performed because of the low number of subjects) in subjects with post-vaccination anti-TT concentrations ≥1.0 IU/ml (3,581.8 [95 % CI 2,211.4; 5,801.4] for serogroup A, 3,891.4 [1,605.7; 9,430.6] for serogroup C, 3,750.9 [1,985.8; 7,084.8] for serogroup W-135 and 7,600.5 [4,588.7; 12,589.3] for serogroup Y) and was lower in subjects in whom the anti-TT concentration remained <0.1 IU/ml after vaccination (1,147.8 [891.5; 1,477.7] for serogroup A, 2,101.0 [1,510.1; 2,923.0] for serogroup C, 1,045.1 [788.0; 1,385.9] for serogroup W-135 and 1,831.1 [1,441.2; 2,326.6] for serogroup Y) (Supplementary table 3). BODY.REACTOGENICITY AND SAFETY: Incidences of local and general solicited symptoms were very low in both groups. In the MenACWY-TT group pain was reported by six subjects (2.3 % [95 % CI 0.8; 4.9]) and redness and swelling each by three subjects (1.1 % [0.2; 3.3]). No local symptoms were reported by subjects in the MenPS group. No local symptoms of grade 3 intensity were reported in either group. Fatigue was reported by five subjects in the MenACWY-TT group (1.9 % [95 % CI 0.6; 4.3]) and no subjects in the MenPS group. Headache was reported by eight subjects (3.0 % [1.3; 5.9]) in the MenACWY-TT group and by two subjects (2.2 % [0.3; 7.9]) in the MenPS group. Fever (defined as axilliary temperature ≥37.5°C) was reported by six subjects (2.3 % [0.8; 4.9]) in the MenACWY-TT group and by one subject (1.1 % [0.0; 6.1]) in the MenPS group. All fever episodes were ≤38.0°C except one, which was >38.0°C and one >38.5°C in the MenACWT-TT group. No subject in either group reported gastrointestinal symptoms during the solicited follow-up period. No grade 3 local or general symptoms were reported by either group, and no solicited symptom led to a medically-attended visit. On observing the low incidences of adverse reactions after vaccination, the investigator re-questioned the subjects before the database was unblinded and the statistical analysis performed, and additional adverse events were not identified. One serious adverse event was reported during the study (reported above). No cases of new onset of chronic disease were reported during the 31-day follow-up period. BODY.DISCUSSION: This is the first study to evaluate quadrivalent MenACWY-TT in an elderly population. MenACWY-TT was immunogenic in this age group, with at least 93.2 % of vaccinees achieving rSBA titers ≥1:128 after vaccination, at least a 13.3-fold increase in post-vaccination rSBA GMTs, and a VR observed in at least 76.6 % of subjects. In contrast with previous studies conducted in children, adolescents and adults, we observed lower responses in the MenACWY-TT group compared to the MenPS group in terms of VR (serogroup A) and GMTs (serogroups A and C), mainly because of a lower response in subjects with high pre-existing rSBA titers (≥1:128), indicative of previous vaccination or exposure, who received MenACWY-TT. Immune hyporesponsiveness, in which the individual fails to mount an immune response after booster vaccination of at least the same, or of a greater magnitude, than that achieved after primary vaccination [16], has been well described in all age groups following repeated vaccination with serogroup C polysaccharide vaccine [17, 35]. Available data from studies in adolescents and young adults using MenACWY-TT or MenACWY-DT (diphtheria toxoid) show attenuated responses to all serogroups in subjects previously vaccinated with polysaccharide vaccines compared to vaccine-naïve subjects receiving their first conjugate vaccine dose [36–38]. This suggests that conjugate meningococcal vaccines only partially reverse immune hyporesponsiveness induced by prior meningococcal polysaccharide vaccination. Nevertheless, immune responses following meningococcal conjugate vaccination in previously vaccinated subjects were consistently high, with seroprotective rSBA levels achieved in the majority of subjects [36–38]. We observed comparable responses regardless of previous meningococcal vaccination in the MenACWY-TT group, except for the lower point values for serogroup W-135 rSBA titers. This study was not designed to evaluate booster responses in previously primed individuals, and we are unable to reach conclusions on the nature of the responses observed. Further studies are needed to define how meningococcal polysaccharide vaccination influences subsequent conjugate vaccination in adults. Our study is one of the few that provide information on the immunogenicity of meningococcal conjugate vaccination in the elderly. ACWY-CRM197 was observed to be immunogenic in older individuals in a study that included a cohort of previously unvaccinated subjects between 55 and 65 years of age with SBA titers (human complement source) that were higher following conjugate vaccination than after meningococcal polysaccharide vaccination [20]. MenACWY-TT was also immunogenic in all four vaccine serogroups in subjects whose age ranged between 56 and 103 years. Our study comprised more females than males, which is typical of the gender distribution of this age group [39]. Conjugate vaccines using TT as carrier are typically highly immunogenic, in part related to T-cell help induced by the TT carrier [40]. We observed a very low percentage of subjects with anti-TT concentrations ≥0.1 IU/ml prior to vaccination and low anti-TT responses to a single dose of MenACWY-TT, consistent with vaccination practices in Lebanon where no tetanus booster is routinely recommended after childhood. Similarly low anti-TT seroprotection rates have been reported in other countries: In Turkey, only 15.4 % of nursing home residents (mean age 71 years) had anti-TT antibodies ≥0.1 IU/ml by ELISA [41]; in Egypt only 9.7 % of elderly individuals had anti-TT antibodies ≥0.15 IU/ml by ELISA [42], while in Spain, only 7.7 % of individuals over 70 years of age had protective titers by hemagglutination [43]. This is in contrast to studies done with MenACWY-TT in other adult populations where pre-vaccination anti-TT concentrations were higher (e.g., 51.5 % with anti-TT ≥0.1 IU/ml prior to vaccination in an Asian adult population aged 18–55 years, including adults from Lebanon [21]). The TT component of MenACWY-TT was highly immunogenic in that study (percentage of participants with anti-TT ≥0.1 IU/ml increased to 79.4 %) [21, 30]. Although vaccination records were not available, the low responses observed in our study may reflect a primary immune response, as observed following a single dose of TT in adults with an unknown or distant history of TT vaccination [44]. The absence of pre-existing immunity to TT or a vaccine response to the TT component of MenACWY-TT may have reduced the available T-cell help, resulting in lower rSBA responses than previously observed, appearing to particularly affect serogroups A and C. This is supported by the observation that rSBA responses were 1.9- to 4-fold higher in individuals who mounted a booster response to TT (concentrations ≥1.0 IU/ml after vaccination) compared with those in whom the magnitude of the post-vaccination anti-TT concentration was lower. Immunogenicity of conjugate vaccines using DT and CRM197 carrier proteins is known to need prior DT priming to generate optimal responses [40]. Our results support the immunogenicity of MenACWY-TT in TT-unprimed individuals, but suggest that effective TT-priming may be critical to enhance the immune response to subsequent TT conjugate vaccines. Studies of meningococcal conjugate vaccines specifically evaluating the effects of carrier priming and of prior polysaccharide exposure on subsequent responses to conjugate vaccination are not available. It is not clear whether the results of our study are predictive for individuals living in other countries where TT boosters may be more frequently administered and where fewer subjects may have received prior meningococcal polysaccharide vaccine. It is possible that in these settings higher immune responses to MenACWY-TT would be achieved in older adults. However, this needs to be confirmed in studies conducted outside of the Middle East. Antibody persistence and the response to subsequent MenACWY-TT doses in the elderly warrant further investigation. It would also be informative to study the immunogenicity of TT boosters in older Lebanese adults given the relatively poor response to the TT component of MenACWY-TT observed in this study. Both of the meningococcal vaccines were associated with very low rates of local and general symptoms. This is consistent with other studies conducted in older individuals where substantially lower reactogenicity has been reported compared to administration of the same vaccines to younger age groups [45]. This study was potentially limited by its open design and because numerous exploratory statistical comparisons were performed without adjustment for multiplicity. Therefore, statistical findings should be interpreted with caution. Inclusion of adults who had previously received meningococcal vaccine was another potential limitation, although we performed sub-analyses to attempt to account for any effects of prior vaccination on the immune response. BODY.CONCLUSION: This study provides the first information on MenACWY-TT immunogenicity and safety in the elderly. In adults 56 years of age and older with a varied meningococcal immunization history, MenACWY-TT was immunogenic, with 93 % of subjects achieving rSBA titers well above the seroprotective threshold (≥1:128) against all four serogroups after a single dose. MenACWY-TT was associated with few minor adverse events. NIMENRIX and MENCEVAX are trademarks of the GlaxoSmithKline group of companies. BODY.ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material 1 (PDF 46 kb) Supplementary material 2 (PDF 49 kb) Supplementary material 3 (PDF 46 kb)

TITLE: Comparison of switching bipolar ablation with multiple cooled wet electrodes and switching monopolar ablation with separable clustered electrode in treatment of small hepatocellular carcinoma: A randomized controlled trialSwitching bipolar vs. switching monopolar ablations ABSTRACT.OBJECTIVE: A randomized controlled trial was conducted to prospectively compare the therapeutic effectiveness of switching bipolar (SB) radiofrequency ablation (RFA) using cooled-wet electrodes and switching monopolar (SM) RFA using separable clustered (SC) electrodes in patients with hepatocellular carcinomas (HCCs). ABSTRACT.MATERIALS AND METHODS: This prospective study was approved by our Institutional Review Board. Between April 2014 and January 2015, sixty-nine patients with 74 HCCs were randomly treated with RFA using either internally cooled-wet (ICW) electrodes in SB mode (SB-RFA, n = 36) or SC electrodes in SM mode (SM-RFA, n = 38). Technical parameters including the number of ablations, ablation time, volume, energy delivery, and complications were evaluated. Thereafter, 1-year and 2-year local tumor progression (LTP) free survival rates were compared between the two groups using the Kaplan-Meier method. ABSTRACT.RESULTS: In the SB-RFA group, less number of ablations were required (1.72±0.70 vs. 2.31±1.37, P = 0.039), the ablation time was shorter (10.9±3.9 vs.14.3±5.0 min, p = 0.004), and energy delivery was smaller (13.1±6.3 vs.23.4±12.8 kcal, p<0.001) compared to SM-RFA. Ablation volume was not significantly different between SB-RFA and SM-RFA groups (61.8±24.3 vs.54.9±23.7 cm3, p = 0.229). Technical failure occurred in one patient in the SM-RFA group, and major complications occurred in one patient in each group. The 1-year and 2-year LTP free survival rates were 93.9% and 84.3% in the SB-RFA group and 94.4% and 88.4% in the SM-RFA group (p = 0.687). ABSTRACT.CONCLUSION: Both SB-RFA using ICW electrodes and SM-RFA using SC electrodes provided comparable LTP free survival rates although SB-RFA required less ablations and shorter ablation time. BODY.INTRODUCTION: Radiofrequency ablation (RFA) is currently accepted as the treatment of choice for patients with very early stage or early stage hepatocellular carcinoma (HCC) when liver transplantation or surgical resection is not feasible [1–4]. According to a recent systematic review, RFA was shown to provide similar quality-adjusted life expectancy for very early HCCs (single nodule <2 cm) in Child–Pugh Class A patients at a lower cost compared with surgical resection [5]. However, although RFA provided comparable survival rates to surgical resection in patients with small HCCs (<3 cm), higher local tumor progression (LTP) rates have also been reported [6, 7]. Therefore, the creation of a sufficient ablative margin (>5 mm) around the target tumor is recommended in order to lower the LTP after RFA [8, 9]. Yet, in clinical practice, there is substantial technical difficulty in covering the entire tumor volume with a sufficient ablative margin using a single electrode, as it requires multiple overlapping ablations [10]. Under ultrasound guidance, repositioning the electrode for overlapping ablations carries technical complexity owing to gas bubble formation, thereby increasing procedure times and the potential of complications [11]. To overcome these limitations, multiple-electrode RFA approaches using switching monopolar, or multipolar RF energy delivery modes have been investigated, and several studies have demonstrated that they indeed provide better local therapeutic efficacy than the single electrode approach [12–15]. As of now, a monopolar RFA system is most commonly used for image-guided thermal ablation [10, 16]. In principle, with bipolar RFA, the electrical current flows between a pair of electrodes and a higher current density is maintained between electrodes [17, 18]. This electrophysiological feature of the bipolar RF delivery mode can allow rapid heating as well as less perfusion-mediated heat sink effect [10, 19, 20]. Recently, switching bipolar (SB) or multi-bipolar RFA using multiple electrodes was demonstrated to create large, regular ablation zones, potentially resulting in lower local tumor progression rates than switching monopolar (SM)-RFA in preclinical and clinical studies [20–24]. This may be attributable to the better heat production efficiency of bipolar RFA at any given current level compared to monopolar RFA[17]. However, despite of the potential benefit of SB- or multi-bipolar RFA in creating a larger ablation zone within a shorter ablation time all in a single session, there has not been any study performed prospectively in the clinical setting. Thus, the purpose of this randomized clinical trial was to prospectively compare, in a random fashion, the therapeutic effectiveness and safety of SB-RFA versus those of SM-RFA in patients with HCCs. BODY.MATERIALS AND METHODS: This study received a research grant from RF Medical Co. (Seoul, Republic of Korea). All authors had complete control of all the data and information submitted for publication at all times. BODY.STUDY POPULATION: The institutional review board of Seoul National University Hospital approved this prospective, randomized single center study (#1310-051-526), and written informed consent was obtained from all patients. This study was conducted by Seoul National University Hospital, Korea and was additionally registered at ClinicalTrials.gov (NCT02675894). Although registration before patient enrollment was recommended, we did not register this study at the beginning, but to declare our study for the researcher, we registered our study on follow-up period. The authors confirm that all ongoing and related trials for this intervention are registered. As far as we know, this study was the first explorative study to therapeutic effectiveness and safety of SB-RFA and SM-RFA. The primary endpoint of the current study was the rate of LTP of the treated lesions, as there have been no studies of direct head to head comparison of two different RFA modes regarding LTP in a randomized clinical trial format. In our institution, approximately 150 patients with HCCs were treated by RFA annually. We assumed that among those patients, 70 patients could be enrolled in this study. And assuming the drop rate as 10%, size of the target population was determined as 77. Between April 2014 and January 2015, 77 patients who met our inclusion criteria below were enrolled in this study. We used the stratified permuted block randomization method on the size of largest HCC in each patient (small (1~2.5 cm) and medium (≥2.5 cm) HCC) with the fixed block size of 4. Patients were randomly assigned (1:1) into the SB-RFA group and SM-RFA group. Randomization process was performed using electrically generated random numbers and managed by our medical research collaboration center. Our inclusion criteria were as follows: (1) pathologic or typical imaging-based diagnosis of HCC; (2) dynamic CT or MRI scan within 60 days prior to RFA; (3) no more than three tumors, with with a diameter ranging from 1.0 cm to 5.0 cm; (4) Child–Pugh A or B liver function status; (5) no contraindication to conventional RFA such as uncooperative patients, intractable ascites, and uncorrectable coagulopathy bleeding; (6) absence of extrahepatic metastases on CT or MRI scan prior to RFA; (7) patients whose ages ranged from 20 to 75 years old; (8) no interventional treatment for hepatic index tumors such as transarterial chemoembolization, percutaneous RFA, or ethanol injection prior to RFA; and (9) patients who are going to undergo RFA for curative purposes. Patients were kept blinded to which group were allocated to, i.e. the SB-RFA group or SM-RFA group. In addition, outcome assessors were also kept blinded to the allocated treatment group. Among the 77 enrolled patients, 8 patients were excluded from the study for the following reasons: (a) patients withdrew their informed consent prior to RFA treatment (n = 4); (b) LN metastasis diagnosed on immediate post-RFA CT scan (n = 1); (c) previous treatment for the index tumor revealed after RFA treatment (n = 2); and (d) biopsy confirmed cholangiocarcinoma (n = 1). Finally, 69 patients with 74 HCCs comprised our study population. Thirty-three patients with 36 HCCs and 36 patients with 38 HCCs were included in the SB-RFA and SM-RFA groups, respectively (Fig 1). Additionally, tumors were subclassified according to tumor size into small (1~2.5 cm) and medium (≥2.5 cm) HCC groups for subgroup analyses. Baseline characteristics of all of the study patients are summarized in Table 1. 10.1371/journal.pone.0192173.g001Fig 1(A) Study protocol of the RFA procedure and intervals between the pre-RFA study, treatment, and follow-up. (B) Flowchart showing the consequences of the study flow. RFA, radiofrequency ablation; HCC, hepatocellular carcinoma; SB, switching bipolar; SM, switching monopolar, F/U, follow-up. 10.1371/journal.pone.0192173.t001Table 1BODY.BASELINE CHARACTERISTICS OF 69 PATIENTS WITH HCCS TREATED WITH RADIOFREQUENCY ABLATION.: OverallSB-RFASM-RFAp-valueM/F ratio 52/17 24/9 28/8 0.781 Age (mean, range, years) 61.4 (33~75) 60.3 (33~75) 62.4 (40~75) 0.36 Child-Pugh: A/B 68/1 33/0 35/1 1.000 Tumor size 2.00 ± 0. 69 1.99 ± 0.54 2.02 ± 0.42 0.894 Etiology of HCC(HBV/HCV/alcoholic/none) 52/9/4/4 27/3/1/2 25/6/3/2 0.655 Number of tumors(single/two) 64/5 30/3 34/2 0.275 Serum AFP(mean ± SD, ng/mL) 25.7 ± 67.1 38.4 ± 88.5 12.9 ± 31.1 0.125 Serum PIVKA(mean ± SD, ng/mL) 30.9 ± 32.6 33.0 ± 42.5 28.8 ± 19.3 0.632 Note.—SM = switching monopolar, SB = switching bipolar, RFA = radiofrequency ablation, HCC = hepatocellular carcinoma, HBV = hepatitis B virus, HCV = hepatitis C virus, AFP = alpha-fetoprotein, PIVKA = protein induced by vitamin K absence/antagonist-II 2, 25] or Liver Imaging Reporting and Data System (LI-RADS)[26] (n = 66). HCCs were classified as perivascular tumors, if the index tumor had any contact with the first- or second degree branches of a portal or hepatic vein that was 3 mm or greater in axial diameter [27]. BODY.RFA PROCEDURE: One experienced radiologist (J.M.L) with over 2000 cases of experience in image-guided RFA, performed all RFA procedures with one clinical fellow or senior radiology resident. Intravenous procedural sedation was induced for the ablation procedure using fentanyl citrate (Hana Pharm, Seoul, Korea), Midazolam (Hana Pharm, Seoul, Korea), and Ketamine (Huons, Hwaseong, Kyunggi, Korea) administered by a special nurse anesthetist with continuous monitoring of vital signs. After disinfection of the skin above the upper abdomen, local anesthesia of 2% lidocaine hydrochloride was applied to the region of electrode insertion. The electrodes were percutaneously inserted under ultrasound guidance with a real-time fusion of ultrasonography (US) and CT or MR images. In the SB-RFA group, three 17-gauge ICW electrodes and a multichannel RF generator (M-3004; RF Medical Co., Seoul, Republic of Korea) were used. The ICW electrode used in our prospective study contains two tiny side holes (0.02 mm in diameter) at the active tips in which less than 1% of the perfused normal saline remains within the electrode for electrode cooling which is then infused into tissue through these holes, at a rate of approximately 1 cc/min, (Fig 2). Sterilized, chilled, 0.9% isotonic saline was used for cooling and tissue perfusion. 10.1371/journal.pone.0192173.g002Fig 2BODY.PHOTOGRAPH OF AN INTERNALLY COOLED WET ELECTRODE WITH TWO TINY (0.02 MM) SIDE HOLES IN THE ACTIVE TIP.: In the SM-RFA group, a separable clustered electrode with three internally cooled electrodes (Octopus RF Electrode, STARmed, Goyang, Kyunggi, Korea), and another multichannel RF generator (Viva RF System, STARmed) were used. Each generator has a maximum power of 200W and performs automatic switching of RF energy among the three electrodes according to impedance changes in an electrode or a pair of electrodes. In both groups, the tip temperature of the electrodes was maintained at a range of 10–20°C by perfusing chilled 0.9% isotonic saline in the electrodes using peristaltic pumps (RFP-300; RF Medical Co., Seoul, Korea and VIVA Pump; STARmed, Goyang, Korea). The prices of all RFA electrodes are set at the same price, according to the insurance policy of Korea, so the treatment cost of approximately 2000$ for each patient was same in both treatment groups. We chose the length of the active tip among 2.0 cm, 2.5cm, and 3.0cm according to the tumor size, shape, location and adjacent large vessels. If necessary, artificial ascites using a 5% dextrose solution was instilled to prevent adjacent organ injury to the subcapsular tumor or to improve the sonic window for the tumor not clearly visible on planning ultrasonography. For placement of the electrode in the target tumor, and for monitoring of the RFA procedures, real-time US-CT/MR fusion imaging systems (eSie Fusion: Acuson S3000, Siemens Healthcare, Erlangen, Germany; PercuNav: EPIQ 7, Philips, Best, Netherland) were used. In general, each of the three electrodes was placed in a triangle formation with an interelectrode distance of 1.5~2 cm. If a sufficient peritumoral margin was not created around the index tumor after one session of RF energy delivery for 8~12 minutes, additional ablations were performed after repositioning the electrodes. The RFA procedure was terminated when the hyperechoic ablation zone was considered to be sufficiently larger than the index tumor on US-CT/MR fusion imaging (Fig 3) [28]. Technical parameters such as the number of ablations, ablation time and total delivered energy were recorded for each target lesion. Additionally, a room occupying time, the time from patient in to patient out for the immediate post-RFA imaging study, was also recorded. 10.1371/journal.pone.0192173.g003Fig 3BODY.HCC IN A 62-YEAR-OLD MAN.: (A) Axial MR images during arterial phase, portal phase and hepatobiliary phase after administration of gadoxetic acid show a 2.6 cm HCC with definitive arterial hypervascularization, venous washout, and hepatobiliary phase hypoenhancement. (B) Real-time US/MRI fusion image before ablation shows a slightly hyperechoic HCC on US image with virtual tumor margin and two electrodes (arrows) placed in the tumor and in the peritumoral area, respectively. (C) PostRFA US/MRI fusion image demonstrates that the virtual tumor margin suggesting the tumor location is covered by hyperechoic ablation zone with sufficient peritumoral margins. (D) Axial (left) and Coronal (right) immediate post-RFA CT images show complete ablation of the target tumor with sufficient peritumoral margins. BODY.EVALUATION OF TREATMENT SUCCESS, EFFICACY, AND COMPLICATIONS: BODY.TREATMENT SUCCESS AND EFFICACY: Immediately after RFA procedures, all patients underwent contrast-enhanced liver CT including arterial and portal venous phases in order to evaluate the technical success of the procedure and development of any possible complications. Technical success was defined as complete coverage of the index tumor by the ablation zone without any residual unablated tumors on immediate follow-up liver CT [10]. If technical success was not achieved, an additional RFA session was done immediately. Complete ablation with achievement of a sufficient peritumoral margin (>5 mm) was determined by the one of the authors with 6 years of experience in clinical imaging (W.C.) using a prototype temporal registration software (HepaCare, Siemens Healthcare) which performs non-rigid registration between the pre-RFA images (CT or MRI scans) and post–RFA CT images [9]. Technique efficacy was defined by the absence of nodular enhancement within or around the ablation zone on 1-month follow-up CT after the RFA procedure [10]. BODY.MEASUREMENTS OF ABLATION SIZE AND VOLUME: Referencing previous studies [29, 30], the diameters and volume of the RFA-induced ablation zone were evaluated on immediate post-RFA contrast-enhanced, multiphasic liver CT scans which included unenhanced, arterial, portal venous, and 3-min delayed phase images. Maximum (Dmx), minimum (Dmi) and vertical (Dv) diameters were measured on the axial and coronal images of the portal phase which showed the largest ablation area showing no contrast enhancement. The volume of the non-enhancing ablation zone on CT was calculated with the assumption of the ablation zone as an ellipsoid using the following formula: ablation volume = π(Dmx x Dmi x Dv)/6 [10, 19, 31] BODY.COMPLICATIONS: All procedure-related complications were recorded, and classified into major and minor complications according to the Society of Interventional Radiology Guidelines [10, 16]. In addition, any additional treatments given to the patients to manage the complications were also recorded. BODY.EVALUATION OF LTP, INTRAHEPATIC DISTANT RECURRENCE, AND EXTRAHEPATIC METASTASES: To evaluate the development of LTP or intrahepatic or extrahepatic metastases (EM), contrast-enhanced multiphase liver CT or MR was performed in all patients every 3~4 months following RFA treatment. Tumor recurrence was assessed in cases which achieved technical success and technique efficacy. The primary endpoint was LTP rate in both treatment groups. LTP was defined as the appearance of any new tumor foci showing arterial enhancement and washout on the portal or delayed scans at the site of the original tumor or adjacent to the ablation zone at follow-up scans [10, 16, 32]. Intrahepatic distant recurrence (IDR) was defined as the emergence of new HCCs in the liver which were not adjacent to the treated site [33, 34]. BODY.STATISTICAL ANALYSIS: All statistical analyses were performed using a statistics software IBM Statistical Package for Social Sciences 24.0 version (Chicago, IL, USA) and Excel 2016 version (Redmond, WA, USA). Continuous variables that could not pass the Shapiro-Wilk normality test were compared using the Z-test or Mann-Whitney test according to whether the number of each group was larger than 30 or not. Other continuous variables were compared using the t-test with unequal variances and Fisher’s exact test was used for the comparison of categorical variables. For the RFA parameters and ablation volumes, adjusted P values were calculated using Holm-Bonferroni Method to correct for multiple tests. The cumulative incidences of LTP, IDR and EM at 6, 12, and 24 months were evaluated using the Kaplan-Meier method with the log-rank test. A P value of less than .05 was considered to indicate a significant difference. BODY.RESULTS: BODY.TECHNICAL PARAMETERS AND ABLATION VOLUMES: The number of ablations was significantly lower (1.72 ± 0.70 vs 2.31 ± 1.37, p = 0.039) and ablation time was significantly shorter (10.9 ± 3.9 vs 14.3 ± 5.0 minutes, p = 0.004) in the SB-RFA group compared with the SM-RFA group. Delivered energy was also lower in the SB-RFA group (Table 2). Mean ablation volume was not significantly different between the two groups (Table 2). In addition, although sufficient peritumoral ablation margin greater than 5 mm was more often achieved in the SB-RFA group, no significant difference was observed (72.2% vs 55.3%, p = 0.153) (Table 2). Room occupying time in the SB-RFA group and the SM-RFA group were 59.7 ± 25.2 vs 64.7 ± 23.4 minutes, respectively (p = 0.393). 10.1371/journal.pone.0192173.t002Table 2BODY.COMPARISON OF RFA VARIABLES AND TECHNIQUE EFFICACY BETWEEN SB-RFA AND SM-RFA GROUPS.: Small HCC (1~2.5 cm)Medium HCC (≥2.5 cm)OverallSB- RFA (n = 25)SM-RFA (n = 26)p-valueSB- RFA (n = 11)SM-RFA (n = 12)p-valueSB- RFA (n = 36)SM-RFA (n = 38)p-valueTumor size 1.58 ± 0.46 1.66 ± 0.40 0.462 2.93 ± 0.40 2.78 ± 0.34 0.342 1.99 ± 0.54 2.02 ± 0.42 0.894 No. of ablations 1.64 ± 0.70 2.23 ± 1.39 0.432† 1.91 ± 1.22 2.75 ± 1.86 0.295† 1.72 ± 0.70 2.31 ± 1.37 0.039‡ Ablation time (minutes) 9.9 ± 4.0 13.0 ±5.0 0.058† 13.2 ± 5.1 17.3 ± 6.5 0.349† 10.9 ± 3.9 14.3 ± 5.0 0.004‡ Energy (kcal) 11.8 ± 6.4 22.2 ± 13.0 0.002† 16.3 ± 3.8 28.0 ± 11.9 0.016† 13.1 ± 6.3 23.4 ± 12.8 <0.001‡ Ablation Volume (cm3) 53.1 ± 19.5 50.6 ± 20.9 0.669 77.9 ± 25.6 64.6 ± 26.3 0.464 61.8 ± 24.3 54.9 ± 23.7 0.229 Technical success 100.0% (25/25) 96.2% (25/26) 1.000 100.0% (11/11) 100.0% (12/12) 1.000 100.0% (36/36) 97.4% (37/38) 1.000 Technique efficacy 96.0% (24/25) 96.0% (24/25) 1.000 100.0% (11/11) 100.0% (12/12) 1.000 97.2% (35/46) 97.3% (36/37) 1.000 Sufficient peritumoral margin (≥5mm) 80.0% (20/25) 57.7% (15/26) 0.132 54.5% (6/11) 50.0% (6/12) 1.000 72.2% (26/36) 55.3% (21/38) 0.153 Major complication 4.0% (1/25) 3.8% (1/26) 1.000 0.0% (0/11) 0.0% (0/12) 1.000 2.7% (1/36) 2.6% (1/38) 1.000 Follow-up(mean ± SD, days) 766.2 ± 211.5 739.0 ± 222.8 0.656 801.5 ± 98.7 751.6 ± 148.5 0.35 777.0 ± 210.4 764.3 ± 170.1 0.448 Note.—SM = switching monopolar, SB = switching bipolar, RFA = radiofrequency ablation, HCC = hepatocellular carcinoma † Adjusted p-values of the Mann-Whitney test using Holm-Bonferroni Method ‡ Adjusted p-values of the Z-test using Holm-Bonferroni Method BODY.TECHNICAL SUCCESS, TECHNIQUE EFFICACY, AND COMPLICATIONS: Among the 74 HCCs, 73 tumors were treated in a single session and one tumor in the SM-RFA group was treated in two sessions. Technical success was achieved in 100% (36/36) of cases in the SB-RFA group and 97.4% (37/38) of cases in the SM-RFA group. A residual tumor was noted on the immediate follow-up CT scan in the technical failure case of the SM-RFA group. The residual lesion was not visualized on planning ultrasonography confidently due to poor sonic windows, and was treated by transarterial chemoembolization (Table 2). Technique efficacy was achieved in 97.2% (35/36) and 97.3% (36/37) of cases in the SB-RFA and SM-RFA groups, respectively. There were no RFA-related deaths observed in this study. Two major complications had occurred including one hepatic abscess requiring percutaneous drainage in the SB-RFA group (2.7%, 1/36) and one active bleeding from an intercostal artery requiring embolization in the SM-RFA group (2.6%, 1/38) (Table 2). BODY.LOCAL TUMOR PROGRESSION, INTRAHEPATIC RECURRENCE, AND METASTASES: BODY.LTP: Mean follow-up periods were 777.0 ± 210.4 and 764.3 ± 170.1 days in the SB-RFA and SM-RFA groups, respectively. No deaths occurred during follow-up. The overall cumulative incidences of LTP at 6, 12 and 24 months were estimated as 0.0%, 6.1%, 15.7% in the SB-RFA group, and 2.8%, 5.6% and 11.6% in the SM-RFA group and were not significantly different between the two groups (p = 0.697) (Table 3, Fig 4). Subgroup analysis according to tumor size also did not reveal any significant differences. There were eight perivascular tumors (8/ 74, 10.8%): five in the SB-RFA group (5/36, 13.9%) and three in the SM-RFA group (3/38, 7.9%) (p = 0.4737). Among them, one case of LTP (1/8, 12.5%) developed in the SB-RFA group. 10.1371/journal.pone.0192173.g004Fig 4Cumulative local tumor progression rates after RFA of (A) overall, (B) small HCCs and (C) medium HCCs. 10.1371/journal.pone.0192173.t003Table 3BODY.LOCAL TUMOR PROGRESSION IN 71 HCCS AFTER SUCCESSFUL RFA.: OverallSmall HCC (1~2.5cm)Medium HCC (≥2.5cm)Months612246122461224SB-RFA 0.0% 6.1% 15.7% 0.0% 9.1% 13.6% 0.0% 0.0% 19.2% SM-RFA 2.8% 5.6% 11.6% 4.2% 4.2% 9.0% 0.0% 8.3% 16.7% p-value 0.697 0.721 0.721 Note.—SM = switching monopolar, SB = switching bipolar, RFA = radiofrequency ablation, HCC = hepatocellular carcinoma BODY.IDR AND EM: Among the 66 patients who achieved treatment success, IDR developed in 9 (9/32, 281%) and 17 patients (17/34, 50.0%) in the SB-RFA and SM-RFA groups, respectively. The estimated cumulative incidences of IDR at 6, 12 and 24 months were estimated as 9.5%, 19.2%, 25.6% in the SB-RFA group, and 8.8%, 29.4% and 50.5% in the SM-RFA group (p = 0.114) (Fig 5). Extrahepatic metastasis occurred in only one patient of the SM-RFA group, 715 days after RFA treatment. 10.1371/journal.pone.0192173.g005Fig 5BODY.CUMULATIVE INTRAHEPATIC DISTANT METASTASIS AFTER RFA.: BODY.DISCUSSION: Our study demonstrated that both SB-RFA using ICW electrodes and SM-RFA using SC electrodes provided comparable LTP free survival rates although SB-RFA required fewer ablations and a shorter ablation time. Previous preclinical studies had demonstrated that SB-RFA could create a larger ablation volume at any given time compared to SM-RFA, and it was expected that SB-RFA would achieve an adequate peritumoral ablation margin and therefore would have a lower LTP compared with SM-RFA [17, 20]. However, according to our study results, the rates of achieved sufficient ablation margins around the target tumor were not significantly different between the two groups. The discrepancy between previous preclinical studies and our human study could be attributed to the use of a real-time US-CT/MR fusion imaging system for in situ monitoring of the ablation zone during RFA procedures in our study. With the use of a real-time US-CT/MR fusion imaging system, we could better monitor the three-dimensional relationship between the echogenic ablation zone and the index tumor during the RFA procedure, which would be difficult to achieve with B-mode US [35]. Thus, the operator was able to terminate the procedure when the echogenic ablation area covered the index tumor with greater confidence. As a consequence, even if SB-RFA could create a larger ablation zone per any given time compared to SM-RFA, the operator may terminate the procedure when the ablation zone was deemed sufficiently larger than the index tumor. Therefore, the ability to secure a sufficient peritumoral margin could be more dependent on the accuracy of the fusion imaging system rather than the difference of efficiency in creating larger ablation zones according to RF energy delivery mode, which probably would also lead to no significant difference in LTP. This is somewhat similar to previous meta-analysis results showing that both microwave ablation and RFA in the management of HCC had similar 1–5 year overall survival, disease-free survival, local recurrence rates and adverse events, although microwave ablation should have clear advantages over RF ablation, such as an improved convection profile, higher intratumoral temperatures, faster ablation times, larger ablation volumes, and less susceptibility to the heat-sink effect [36]. To the contrary, the shorter procedure time may indeed be beneficial for both patients and for the operator. The technical success rates of our study were 100.0% and 97.4% in the SB-RFA and SM-RFA groups, respectively. All tumors were treated in a single session except for one tumor which had been treated in two sessions. The high technical success rates observed in both of our study groups could be attributed to the better efficiency of the multiple electrode approach, in either switching monopolar or bipolar modes, compared with conventional monopolar RFA using a single electrode in delivering RF energy to the target tissue, thereby allowing the creation of larger ablation zones [19, 29, 31, 37]. Another factor for the high technical success observed in our study could be attributed to the fact that compared with the multiple overlapping ablation approach using a single electrode which has great technical difficulty with US guidance, there was much less demand to relocate the electrode when multiple electrodes were used [38]. However, the cumulative 2 year LTP rates of SB-RFA and SM-RFA were still higher than 10%, which is much higher than the reported results of surgical resection [39]. This could be mainly attributed to the fact that a sufficient peritumoral ablation margin greater than 5 mm in three dimensions was achieved in 72.2% of tumors in the SB-RFA group, and 55.3% in the SM-RFA group. Indeed, the creation of a sufficient peritumoral margin in three-dimensions with RFA was quite challenging in several cases with advanced liver cirrhosis due to the poor sonic window, even under the guidance of a real-time US-CT/MRI fusion system [35]. Furthermore, registration accuracy of real-time US-CT/MRI fusion has been reported to be approximately 3 mm ~5 mm, and therefore, this misregistration could have resulted in the creation of an insufficient peritumoral safety margin [40]. Considering that the creation of an ablative margin of at least 5 mm is widely accepted to be one of the most important factors for reducing LTP in HCC after RFA [8, 39, 41], further improvement of the efficiency of multiple electrode RFA in creating large ablation zones and the development of an accurate in situ monitoring tool to better show the relationship between the index tumor and the ablation zone may further decrease the LTP after RFA. Although bipolar RFA has better heat producing efficiencies, tissue dehydration and charring around the electrodes more commonly occurs in the bipolar mode than in the monopolar mode due to the higher current density around the electrodes, resulting in less efficiency in delivering RF energy to the target tumor [42]. In order to circumvent this problem, we used ICW electrodes for SB-RFA, which allow simultaneous internal cooling and interstitial perfusion of normal saline in our study [20]. Although internally cooled electrodes can preferentially decrease the heating of tissue nearest to the electrode, effectively preventing the charring of tissue in monopolar RFA [16], this technique does not sufficiently prevent overheating of the tissue around the electrode in the bipolar mode, which requires rapid switching of the active electrodes or elongation of the RF ablation time [23, 24]. In this regard, additional infusion of saline into the tissue can increase the electrical conductivity at the electrode-tissue interface and further decrease the heat generated by the electrical current flow through the tissue with high electrical impedance [43–46]. Furthermore, it can also increase thermal conductivity and the heat is more easily carried away from the electrode [17]. Our results of a lesser number of required ablations and shorter ablation times are largely owed to this ability of large and regular ablations using SB-RFA with ICW electrodes. As for adverse events, both SB-RFA using ICW electrodes and SM-RFA using separable clustered electrodes showed low rates of complications (< 3%), similar to the results (4.1%) of a previous systematic review [47]. Theoretically, complication rates related to the RFA procedure can be increased in accordance with the number of electrode insertions, but in our study, safe and precise planning of the electrode insertion route, taking care to avoid bile ducts and major vessels, was possible under the guidance of fusion imaging, and the potential increase in complication rates related with the electrodes was able to be minimized. Furthermore, although there were no cases of skin burn in the SM-RFA group, SB-RFA has an advantage of not requiring grounding pads, and therefore, no risk of skin burns. Recently, several studies [22, 48] showed the feasibility of RFA using the “no-touch” ablation technique with multiple electrodes in the multipolar mode, and also reported its promising outcomes including low LTP rates. The multiple electrode approach used in our study, especially in bipolar mode, can allow the “no-touch” ablation technique. In addition, considering that drainage vessels change in HCC from hepatic veins to peritumoral sinusoid or portal veins [49, 50], electrode insertion into the peritumoral area or peripheral portions of the target tumor can induce more extensive thrombosis in the draining peritumoral vessels which would be beneficial in decreasing the risk of intrahepatic metastases through the vessels. Indeed, in our study, the IDR rate was lower in the SB-RFA group, albeit without statistical significance. Experimental evidence that tumorogenic factors facilitating unwanted tumor recurrence may be produced after RFA treatment in the residual tumor and surrounding liver parenchyma have been raised up and these factors could differ in different modes of ablation[51–54]. And this might be another reason for lower IDR rate in the SB-RFA group. This phenomenon should be further evaluated in following studies. There are several limitations to our study. First, this study compared the two groups using two different electrodes and two different RFA modes. Thus, compounding effects could not be completely avoided. Second, we did not perform the sample size calculation, and power analysis for this study and only a small number of patients were enrolled in our study for the explorative purpose. Therefore some results such as the differences in the LTP, rates of complete ablations, number of ablations in the subgroup analysis, and IDR were not confirmed in terms of statistical significance. Further study with a larger number of patients with longer follow-up periods is warranted. In conclusion, both SB-RFA using ICW electrodes and SM-RFA using SC electrodes provided comparable LTP free survival rates although SB-RFA required less ablations and a shorter ablation time. In addition, as there were no clear differences between SB-RFA and SM-RFA, either technique can be favorably used for the percutaneous ablation of HCCs, depending on the operator’s familiarity and preference. BODY.SUPPORTING INFORMATION: S1 FileBODY.CONSORT CHECKLIST.: (DOC) Click here for additional data file. S2 FileBODY.FULL STUDY PROTOCOL IN KOREAN.: (DOC) Click here for additional data file. S3 FileBODY.SUMMARIZED STUDY PROTOCOL IN ENGLISH.: (DOCX) Click here for additional data file. S4 FileBODY.ANONYMIZED RAW DATA.: (XLSX) Click here for additional data file.

TITLE: Optimal dose of dexmedetomidine for sedation during spinal anesthesia ABSTRACT.BACKGROUND: Sedation in spinal anesthesia can reduce patient's anxiety and discomfort. Dexmedetomidine has a sedative, hypnotic, analgesic, and minimal respiratory depression effect. However, use of the dexmedetomidine is associated with prolonged recovery. This study was designed to investigate the optimal dose of intravenous dexmedetomidine for proper sedation with minimal recovery time in spinal anesthesia. ABSTRACT.METHODS: One hundred twenty eight patients, aged 20-70 years (58.8 ± 0.7), were recruited. After performing the spinal anesthesia with hyperbaric bupivacaine (13 mg), a loading dose of dexmedetomidine (1 µg/kg) was administered for 10 min, followed by the maintenance infusion of the following: Group A (n = 33; normal saline), Group B (n = 35; dexmedetomidine 0.2 µg/kg/hr), and Group C (n = 39; dexmedetomidine 0.4 µg/kg/hr). Heart rate, blood pressure, and the bispectral index score (BIS) were recorded during the operation. In the recovery room, modified aldrete score (MAS) was measured. ABSTRACT.RESULTS: There were no significant differences in mean blood pressure and heart rate among the three groups. BIS was not significantly different among the three groups from baseline to 60 min after the infusion of dexmedetomidine. BIS were significantly increased in Group A after 70 and 80 min, and Group A and B after 90, 100, 110 min of dexmedetomidine infusion (P < 0.05). MAS was higher in Group A as compared to Group B and C, within 30 min after admission in the recovery room (P < 0.05). ABSTRACT.CONCLUSIONS: The loading dose (1 µg/kg/10 min) of dexmedetomidine was sufficient for surgery of less than 60 min. Dexmedetomidine infusion followed by maintenance dose (0.2 µg/kg/hr) was sufficient for surgery within 90 min. BODY.INTRODUCTION: Spinal anesthesia has the advantage of being able to maintain spontaneous breathing as well as relaxing the necessary muscles for surgery. It also has the advantage of being free from the risks of intubation and pulmonary aspiration. However, the time limit and patients' anxiety of spinal anesthesia present disadvantages. Generally, most failures in regional anesthesia, including spinal anesthesia, are related to inadequate sedation and relief of anxiety rather than technical problems. Adequate sedation in spinal anesthesia relieves the anxiety of the patient, improves physiological and psychological stress, and increases the satisfaction of both the surgeon and patient [1,2]. In spinal anesthesia, propofol and midazolam are commonly used for sedation. Propofol produces rapid and deep decline in the level of consciousness. With a continuous infusion of propofol, the infusion rate can be adjusted to reach the desired level of sedation. Regardless of the length of infusion duration, rapid recovery is seen once infusion is discontinued. However, the use of propofol often leads to a decline in cardiovascular function and respiratory function [3-5]. Although midazolam is a lipid-soluble benzodiazepine with a rapid onset and recovery time, the onset time of midazolam is slower than propofol. Also its clearance time when infused at a high dose is slower than that of propofol. Moreover, midazolam causes a slight drop in blood pressure and mild to moderate respiratory depression [6]. Dexmedetomidine, a selective alpha2-adrenergic agonist, affects the locus caeruleus area, which is related to the modulation of sleep regulation and respiratory control, and has a sedative effect. The analgesic effect is due to the activation of the alpha2-adrenergic receptors of the central nervous system [7,8]. Dexmedetomidine reduces biological stress responses and reduces the heart rate and blood pressure to moderate levels by lowering catecholamine secretion. It also has minimal or no effect on the respiratory rate and tidal volume [9]. However, the use of dexmedetomidine can cause prolonged recovery [10,11]. The use of dexmedetomidine as a sedative in regional anesthesia, such as in epidural anesthesia, spinal anesthesia, and nerve blocks, shows a much greater sedation in the post anesthetic care unit (PACU) compared to the use of propofol [12]. In patients of 65 years and over who had spinal anesthesia with a low dose of bupivacaine, the use of dexmedetomidine had a longer PACU stay than the control group that used normal saline [13]. The present study investigated on the optimal dose of dexmedetomidine with minimal recovery time which gave adequate sedation for spinal anesthesia within 1-2 hr. BODY.MATERIALS AND METHODS: One hundred and twenty eight patients, aged 20-70 years, of American Society of Anesthesiologists physical status I and II, were recruited. An anesthesiologist visited the patients who planned to have elective surgery under spinal anesthesia the day before the surgery. Informed consent about the spinal anesthesia and sedation during surgery was obtained. Patients were excluded from the study if they had arrhythmia, heart failure, renal failure, liver failure, or bleeding tendency. Premedication was not performed. After arrival in the operating room, noninvasive blood pressure, heart rate, oxygen saturation, and bispectral index (BIS) monitoring were performed. Before spinal anesthesia, crystalloid 10 ml/kg was administered intravenously as prehydration. The patient was placed in the lateral recumbent position and a spinal tap in L 3/4 was performed. Hyperbaric bupivacaine 13 mg was administered intrathecally. The patient was then placed in the supine position, and oxygen 3 L/min was administered. After the confirmation of successful spinal anesthesia, dexmedetomidine loading dose 1 µg/kg was administered intravenously for 10 min. With different syringes, the maintenance infusion was administered intravenously (Group A: normal saline, Group B: dexmedetomidine 0.2 µg/kg/hr, and Group C: dexmedetomidine 0.4 µg/kg/hr). The intravenous administration of dexmedetomidine was discontinued once the surgeon began to suture the surgical site. Three types of syringes were prepared on 50 ml syringes (Group A: saline 50 ml, Group B: a 50 ml mixture of saline 49 ml and dexmedetomidine 100 µg, and Group C: a 50 ml mixture of saline 48 ml and dexmedetomidine 200 µg). The syringes were prepared in advance by nurses and were chosen by using a table of random numbers. Noninvasive blood pressure, pulse oxymetry, and BIS were monitored in all patients before surgery, immediately after ending the loading dose of dexmedetomidine, and at 10 min intervals thereafter. In the PACU after surgery, assigned nurse recorded modified aldrete score (MAS). The adequate sedation level for surgery was set at mild to moderate, at BIS 60-80. In the PACU, MAS was assessed, which consisted of activity, breathing, circulation, consciousness, and oxygen saturation. The recovery was considered prolonged, when the MAS was under 9 (out of 10), and the duration of stay was longer than 30 min in the PACU. Atropine 0.5 mg was administered intravenously when bradycardia (heart rate below 50 beats/min) occurred. Ephedrine 5 mg and crystalloid 300 ml was administered intravenously when hypotension (mean arterial pressure 50 mmHg or below) occurred. The patients with the surgery lasting more than 2 hr 30 min (Group A = 4, Group B = 3, and Group C = 4), and were awake in need of additional sedatives were excluded from the study (Group A = 4, Group B = 4, and Group C = 2). The final remaining number of patients were 33 in Group A, 35 in Group B, and 39 in Group C (Fig. 1). All data were recorded as mean ± standard deviation. We used Statview 5.0 (SAS institute, Cary, USA) for statistics. When BIS had a difference of 15 or more, α value was set at 0.05, and β value was set at 0.2, and the sample size was set at 27 (before starting the intravenous administration of dexmedetomidine, initial BIS was 94.1 ± 3.5). ANOVA was used to analyze continuous variables, Scheffé's test for posthoc testing, χ2 test on individual patients' statistics, and the Kruskal-Wallis test for MAS in the PACU. P value of 0.05 or below was considered statistically significant. BODY.RESULTS: There were no significant differences among the three groups in the patient's gender, age, weight, and operating time (Table 1). BIS started to decline after ending the administration of the loading doses in all groups. There were no differences in BIS among the three groups from the point of administration of the loading dose of dexmedetomidine for 60 min. There were significant differences at 70 min (Group A: 82.2 ± 8.2 min, Group B: 69.2 ± 16.1 min, and Group C: 69.1 ± 11.6 min) and at 80 min (Group A: 82.0 ± 7.6 min, Group B: 72.5 ± 15.4 min, and Group C: 73.3 ± 11.0 min). Group A was significantly different compared to Group B and C (P < 0.05): at 90 min (Group A: 86.5 ± 9.2 min, Group B: 85.1 ± 9.6 min, and Group C: 71.7 ± 11.6 min); at 100 min (Group A: 86.6 ± 7.8 min, Group B: 81.9 ± 8.4 min, and Group C: 69.1 ± 8.3 min); at 110 min (Group A: 90.3 ± 9.3 min, Group B: 83.9 ± 10.4 min, and Group C: 74.6 ± 12.0 min); and at 120 min (Group A: 89.7 ± 8.1 min, Group B: 83.4 ± 11.1 min, and Group C: 78.3 ± 10.2 min). Groups A and B showed significant differences compared to Group C (P < 0.05) (Fig. 2). The heart rate and mean arterial pressure showed a decrease, but there were no significant differences among the three groups (Fig. 3). In the PACU, MAS was significantly high for 30 min in Group A compared to Group B and C (P < 0.05) (Fig. 4). BODY.DISCUSSION: In spinal anesthesia, using no maintenance dose after a loading dose of dexmedetomidine 1 µg/kg brought adequate sedation within 60 min. An infusion of a maintenance dose of 0.2 µg/kg/hr brought adequate sedation within 80 min, and an infusion of a maintenance dose of 0.4 µg/kg/hr brought adequate sedation within 120 min. The distribution half-life of dexmedetomidine is known to be 5 to 10 min. The termination half-life is known to be within 2-3 hr; and it is similar in healthy Koreans. It has been reported that dexmedetomidine has linear pharmacokinetic characteristics and dose-dependent sedation effects [14]. Lee et al. [14] reported that when a loading dose of dexmedetomidine 1 µg/kg for 10 min was administered and infused continuously, on the average, the peak concentration was reached in 17 min, and the termination half-life was 2 hr and 10 min. We believed that the loss of sedative effects occurs at around 60 min after the administration of a loading dose without continuous infusion. It was reported that when an MRI was taken in a pediatric patient who needed the sedation of within 60 min, even with a high dose administration of dexmedetomidine (2-3 µg/kg/10 min loading dose), the adequate sedation was not achieved and additional drugs were needed [15]. However, in the case of spinal anesthesia, the spinal anesthesia itself is reported to have sedative effects and a low loading dose of dexmedetomidine alone is believed to bring adequate sedation [16]. Depending on the dose of continuous intravenous administration after the administration of loading dose, the difference in duration of sedation is believed to be dependent on the aforementioned linear pharmacokinetic characteristics. In this study, the number of patients within 90 min from the loading dose infusion of dexmedetomidine satisfied the sample size, but the number of patients over 90 min from the loading dose infusion of dexmedetomidine was lower than the sample size. As such, a further study is necessary for sedation over 2-3 hr. In general, the Ramsay sedation score and the observer's assessment of alertness and sedation (OAA/S) score are commonly used to assess sedation. However, the disadvantages of these assessment methods are that the sedated patient must be alert for assessment, and that the assessor makes a subjective assessment depending on the patient's response. Moreover, a real time assessment is difficult. BIS monitoring has the advantage that sedation can be maintained without external stimulation, and the objective, real-time assessment is possible. BIS values are correlated to anesthetic depth of inhalational anesthesia and intravenous anesthesia, and also correlated to sedation using propofol and midazolam [17,18]. Liu et al. [19] reported that OAA/S scores and BIS are correlated to the alert state, sedation state, and deep sedation state. Sleigh et al. [20] stated BIS is an index for consistent monitoring of the depth of sleep. In a study comparing BIS and OAA/S in the sedation using dexmedetomidine and propofol, 85% of the patients showed 40-60 of BIS value, a deep sedation state, in the dexmedetomidine group when the OAA/S score was 3, which was a relatively greater sedation score than the propofol group. We set BIS for adequate sedation level at 60-80. Sedation was maintained well except the small number of patients who needed additional sedation during surgery. As such, the sedation using dexmedetomidine, with BIS as the sedation index, showed different results as compared to the study by Kasuya et al. [21] As aforementioned, the sedation effects of spinal anesthesia itself, and the fact that the spinal anesthesia and sedation were induced simultaneously, could induce sedation even at a high BIS value. Relatively low dose of dexmedetomidine can also induce sedation at a high BIS value. Unlikely other drugs, dexmedetomidine affects the locus caeruleus and induces sedation. Its pattern is more similar to sleep than sedation. As aforementioned, it is reported that BIS is related to the depth of sleep, and it may be also applied to the effects of sedation using dexmedetomidine. Further study is required on whether dexmedetomidine has an effect on BIS itself, and the possible dose-dependent difference. Spinal anesthesia is often used for ambulatory surgery. It is necessary to reduce the time of stay in the PACU, and this is especially important for patients who have had spinal anesthesia with sedation. The group that was administered only a loading dose had a MAS which allowed the patient to be discharged at the time of arrival in PACU. However, a low dose of dexmedetomidine was infused in all three groups, and the patients had appropriate MAS for discharge within 30 min. In the PACU, BIS were approximately 90 in all groups, and there was no significance. There is a report that dexmedetomidine used for sedation delays the patients' recovery. In those cases, the loading dose was administered, then a maintenance dose of 0.6 µg/kg/hr or more was used, which was different from the present study [10,11]. However, when dexmedetomidine was used in pediatrics for the sedation for an MRI, it was reported that there was no correlevance between recovery time and dose [22]. Although spinal anesthesia with a low dose of dexmedetomidine was performed in this study, and we did not observe delayed recovery, it is difficult to compare recovery profiles with only simple comparisons in the PACU. The greatest benefit of sedation by dexmedetomidine administration is that it can cause sedation without respiratory depression, and respiratory depression was not observed in our study. In general, the most common side effects of dexmedetomidine are hypotension and bradycardia. When dexmedetomidine is used with spinal anesthesia, it may increase the frequency of such side effects. In this study, there was no case of hypotension that needed rescue drugs; however, bradycardia that needed rescue drugs occurred in 3 patients in Group A, 3 patients in Group B, and 1 patient in Group C. There is a report of bradycardia and hypotension occurring in pediatric patients with a large amount of dexmedetomidine administered [22]. It is likely that the frequency of bradycardia and hypotension does not increase when a low dose of dexmedetomidine is administered with spinal anesthesia. The use of dexmedetomidine causes sedation, but because the patient can be aroused at anytime, it is commonly used in sedation for procedure. The limitation of the present study is that dexmedetomidine was administered after performing spinal anesthesia. When sedation is performed for the purpose of reducing patient anxiety during surgery, it is believed that adequate sedation effect needs to precede the start of the surgery, ideally. As soon as the patient is on the operating table, the infusion needs to be started, and it helps to reduce anxiety from spinal anesthesia itself and dexmedetomidine's relatively long onset time. In conclusion, dexmedetomidine can be useful in spinal anesthesia for sedation. For surgeries within 1 hr, sedation can be induced by only 1 µg/kg/10 min loading dose. For surgeries within 90 min, 0.2 µg/kg/hr maintenance dose can produce sedation without delayed recovery.

TITLE: An investigation on the effect of strength and endurance training on depression, anxiety, and C-reactive protein's inflammatory biomarker changes ABSTRACT.INTRODUCTION:: This study is performed to investigate the effect of strength and endurance training on the levels of depression, anxiety, and C-reactive proteins inflammatory biomarker changes. ABSTRACT.MATERIALS AND METHODS:: The research method was experimental, and the statistical population is formed of 300 volunteer male students. After the pre-test, 120 subjects with notable depression and anxiety levels obtained from Beacke and Ketel's questionnaires were selected and randomly divided into two groups of strength and endurance, each containing 60 subjects, and then, again into two groups of experimental and control, each with 30 subjects. All 120 subjects were blood-sampled in the first stage to determine CRP concentration. After 20 sessions of strength and endurance exercises again depression, anxiety, and C-reactive proteins testes were used for both control and experimental groups. Ultimately, the obtained data were analyzed by using t-test in dependent and independent groups and covariance analysis in P ≤ 0.05 level. ABSTRACT.RESULTS:: The results showed that the average of age is 25.1 ± 3.2, average of weight is 70.4 ± 8.4 and average of height is 169.8 ± 12.1, in the subjects. Also, the strength and endurance training had reduced the anxiety by 27% (P = .0001), depression by 37% (P = .0001) and C-reactive proteins by 20% (P = 0.0001), in the subjects. ABSTRACT.DISCUSSION:: Regarding the different effects of training types on research variables, the results showed that the endurance training has a greater effect in reducing the depression, and strength training, in blood C-reactive proteins reduction, Although, no significant difference was observed between anxiety-reducing effects of strength and endurance training. BODY.INTRODUCTION: The percent epoch is called the stress and psychological pressure age.[1] The results of various researches showed that psychosomatic diseases such as depression and anxiety have a deep effect on personal and social performance and grounds for many physical diseases such as cardiovascular ones. Unfortunately, today in the world, attention to psychological health in families and families and sureties is not as important as physical health, Unaware of the fact that the origin of many physical diseases is in psychological injuries.[23] Most psychologic health professionals believe that all anxiety disorders refer to damaging experiences or previous pressures in life, which are unconsciously oppressed and turned into anxiety. Studies in recent decades concerning the prevalence of this disease show that more that 27% of those referring to psychiatrists are affected by various degrees of anxiety.[4] The intensity is worrisome when crating disorder in normal life, which is usually together with physical symptoms.[56] Another prevalent psychological disorder is depression, which is created by disorders in temper, emotions and reduced biologic force. Negative feelings and helplessness, disoperation and loneliness, feeling of inviolability, guilt, and sorrow in the face, and isolation are obvious symptoms.[78] The research shows that one in the in the society suffers from depression and women are affected as twice as men.[4] Civet the provolone of depression and anxiety in various societies. Numerous researches are performed in treating these diseases and in addition to medication, psychiatry, shock-treatment and cognitive cures, the exercise therapy have been mentioned as an effective technique. Exercise therapy is a novel treatment which increasingly, and relying on the researchers’ conclusions, has drawn public attention and the depressed and anxious patients with negative and despairing mentality toward other treatments. Studies show that the continuous and regular exercising followed by sweating and an increase in the heartbeat could control the blood pressure and emits tranquilizing and pain-relieving hormones such as Endorphin and Encephalin which cause a better reaction in individuals’ neural system in critical conditions and anxieties. Experience brights horizons in their release from psychologic stress relying upon this therapy. The fact that exercise has an important and effective role in curing depression and anxiety is supported by using exercise as a supplement with other cures is far more effective.[910] Of course further studies should be carried out to be concise about the effects of endurance and strength exercises in reducing the depression and anxiety disorders and also in planning an effective sport-therapy protocol. Another variable in the research is C-reactive protein or CRP, a member of proteins family, generated protein or CRP, a member of proteins family, generated following inflammation processes. Following any inflammation, infection and texture irritation, it may increase by l000 times in blood; therefore its measurement has a special value in diagnosis, confirmation and treatment of various diseases such as infection, trauma and inflammation in endothelial cells of coronaries.[711] This protein is quickly disappeared in blood after the inflammatory response has subsided. The research shows significant relation between intense depression and anxiety and increased amount of CRP in men's and women's blood.[1213] Also, significant relation was observed between the increase level of this protein and cardio as cular and brain strokes.[14] Toker and Shiron[15] indicated the relation the relation between depression and anxiety, and CRP, but that differed is each sex. the research conducted by Wiles jus approved the relationship between acute depression with CPR decrease in men[16] on the other hand Douglas and Taylor believe that there is weak relation between depression and anxiety with CPR decrease and CPR is an only indirect and moderato parameter to cause coroner vessels diseases.[12] On the other hand, the importance of physical activity, as a supplementary part of daily life is becoming more obvious and scientific evidence indicate health. Benefits of exercise.[17] The research has shown that physical activity with appropriate frequency, intensity and duration is effective in enhancing the physical and mental health levels.[18] Physical activity, in addition to creating physical fitness and developing motive skills, prevents many diseases such as cardiovascular, hypertension, fatness, arthritis, anxiety, depression to occur.[1920] The psychologists of a depressed person, is that of emptiness and worthlessness and by creating self-rehabilitation and returning self-confidence by exercise, while eliminating negative feelings and creating a “one can change” and “one can be positive and live positively” approach, pave the ground for positive feelings. Through exercise, one feels able to control his/her life even in the hardest situations; his/her life.[14] In this realm, Philips supports the effect of aerobic and non-aerobic exercises on the reduction of youth depression but illness depression in middle age and after are not under the effect of sport exercises.[9] Research show an inverse relation between fitness level and CRP levels in adult's blood, which means that increasing the fitness, the CRP in blood decreases.[14] The bottom-line is that depression and anxiety are among that most prevalent psychological disease with considerable role in cardiovascular disorders. Given the significant relation between anxiety, depression and cardiovascular diseases and CRP concentration in blood; and given the effect of fitness levels in reducing CRP, the efforts of the researchers forward finding suitable solutions to prevent and cure cardiovascular diseases through controlling psychosomatic disorders such as anxiety and depression, have led to presenting numerous clinical and medicinal methods but less attention with these variables at the same time. Therefore, given the confirmed effects of physical activity in physical and psychological health in individuals in different ages[621222324] the authors are going to study the effect of two physical activity types namely, strength and endurance training, on the changes in the levels of depression, anxiety and blood CRP concentration as an indicator of inflammation in cardiovascular system of young men. The previous studies have not examined the effects of physical activity, psychological disease and physiological changes, simultaneously. Therefore, the aim of our study is to investigate the effect of strength and endurance training on the levels of depression, anxiety, and C-reactive proteins inflammatory biomarker changes. BODY.MATERIALS AND METHODS: This experimental investing at ion is performed through comparison of research variables and aiming at studying the effect of strength and endurance training on depression, anxiety and C-reactive protein's inflammation biomarker. In order to choose this study statistical population, there was a call in Azad university physical education organization among male volunteer students. Then 300 volunteer students between 20 to 30 years old were selected in 2011. The statistical population includes 300 young men between 20 to 30 years old, and after depression and anxiety test, a sample of 120 of them with notable depression and anxiety level were selected and randomly divided into two groups of strength and endurance training, each included 60 subjects, and any of these groups too, were divided into groups of 30, one control and one experimental group. In this research, in addition to blood samples, Beacke questionnaire was used to determine the depression and kettle questionnaire, to determine the anxiety levels. To be assured in content narration in the questionnaires, experts’ ideas on the issue were applied. After applying all suggested ideas, the Cronbach Alpha test was performed to determine the questionnaires” internal reliability and the obtained values on the Beacke questionnaire (.74) and Kettle questionnaire (.89) verified the internal correlation. The achieved reliability in this research is in consistent with the values written in the Chegunian (2002) research for Beackes’ depression questionnaire (0.78) and for Kettles’ anxiety questionnaire (0.82) [Table 1]. Table 1Demographic date After selecting sample groups and performing pre-tests, the experimental groups did strength and endurance training during 20 sessions, 1.5 h each for l0 weeks (two sessions/ week) separately. The specific exercises for the strength group included short-distance sprint running, strength (power) work- outs, and working with equipment, and the specific exercises for the endurance group included aerobic warm-up and at least, 1200m running at the beginning and by 2500m gradually to the end of period. After performing the exercises and ending the activity period, the tests for all subjects in both control and experimental groups. In order to analyze the research findings, because of natural distribution and consistent variance of variables, the dependent t-test was used to compare the pretest and after test averages in each group, and the independent t-test, to compare pretest and after test averages between two groups, and covariance analysis, to study the real effect of in dependent variable. The significance level for all tests was considered as P804; 0.05. All the statistical analysis has been accomplished by SPSS/13 software. BODY.RESULTS: Based on the finding in Table 2, observed F in alpha level of 0.05, Concerning the effect of strength and endurance physical activates on the depression levels of the subjects, has been significant in both control and test groups. The obtained factor shows that the depression level of subjects has reduced by ~ 37% under the in fluency of strength and endurance exercises. Table 2Covariance analysis for depression in control and experimental groups in strength and endurance exercises Based on findings in Table 3, the deserved F concerning the effect of strength and endurance physical exercises in control and test groups on the anxiety levels in subjects, has been significant in the level of α = 0.0.5. The obtained factor shows that the strength and endurance training has had an effect of about 27% in reducing the subjects’ anxiety. Table 3Covariance analysis of anxiety in control and test (experimental) groups over strength and endurance exercises Based on findings in Table 4, the observed F concerning the effect of strength and endurance exercises on the subjects’ blood CRP levels was significant in both control and test groups in α = 0.0.5 level. Table 4Covariance analysis of CRP for control and test groups in strength and endurance training In other words, given the obtained factor, the CRP levels in subjects’ blood have reduced by about 20% under the influence of the performed exercises. Based on findings in Table 5, the significance of obtained concerning the differences in the effects of exercise type on the subjects’ depression has more effect in reducing the depression than strength training. Table 5The effect of exercise type on depression, anxiety and CRP levels of experiment groups Also, insignificance of obtained P for anxiety indicates the indifference of exercise type's effect on this variable. For CRP, too, the significance of obtained P, indicates higher effectiveness of strength exercises in reducing this variable. BODY.DISCUSSION AND CONCLUSION: Based on research results about 50% of statistical population suffered from different degrees of depression and anxiety, from among which, 120 persons with medium depression and anxiety levels were selected to form control and anxiety levels were selected to form control and experimental groups the pre-test results in both control and experimental groups showed that the subjects’ depression level was 10.75 by average [Table 2] and their anxiety level, 35.5 [Table 3], which after strength and endurance exercises, in the second test, these values reduced to 7.54 for depression and 29.66 for anxiety in experiment groups. These results support the idea that physical strength and endurance exercise has reduced the subjects’ depression and anxiety. These results are consistent with those obtained by Stewart and Boomsma and De Moor and Phillips, based on the effect of physical exercise on reduction of psychological disorder such as depression and anxiety,[25] there was no agreement on the amount of its effects though. De Moor and Boomsma believe that acute cases of anxiety and depression are less affected by physical exercises’. Also, Wiles and Haase showed that considerable physical activity has positive effect on the individuals’ psychological health.[16] Physical activity, in addition to modifying hormonal mechanisms, with desirable psychological effects such as increased self-confidence, improved self-respect, enhanced environmental and social interactions, reduced focus on negative mental occupations and replacement of undesirable behaviors with an active and dynamic life style, lead to elevated levels of psychological health.[1726] It seems that the specific age conditions of the young research population and their mental preoccupations for ambiguous situations of education, job, marriage, etc are among the emergence factors of depression and anxiety in the research population and anxiety in the research population and participating in continuous activates, has led to reduced depression and anxiety in the subjects by creating desirable behavioral habits and psychological effects. Other findings in covariance analysis showed that 37% of the depression level of the subjects had reduced following strength and endurance training [Table 2], which is consistent with De Moor and Philips’ results based on the effect of continuous physical activity in leisure time on reduced depression a higher effect of endurance training on depression decrease.[925] It is worth mentioning that similar researches concerning the effect of training type on depression the effect of training type on depression the effect of training type on depression levels have been less performed. Also the results of covariance analysis showed that because of strength and endurance training, about 27% of subjects’ depression has decreased. Although, a significant difference was not achieved between the effects of physical activity types of strength and endurance exercises. Some other previous studies results on the different effects of the exercise type were in agreement with the results of this part of the study,[1826] which seems that this lack of agreement was because of the statistical population, the exercise type or the testes’ physical and mental conditions. Other findings showed that blood CRP levels in control and test groups were about 20% higher than normal. The value obtained in the first test for all groups was 1.25 by average and in the second test, 1.001 for test groups Table 5. These findings emphasize the fact that continuous training can considerably lower the blood CRP as an in Flomaton biomarker. These findings are consistent with the results of Toker, Askari and Stewart.[141517] Also, the covariance analysis results regarding the effect of strength and endurance exoduses on the subjects’ blood CRP showed that blood CRP, as an important indicator of cardiac veins inflammation,[27] has reduced by about 20% and the strength training had a higher effect in this reduction. However, Douglass and Taylor believed that CPR only was an intermediate and direct factor in the appearance of coronal diseases. These results are consistent with Askari and - Danesh based on the effect of physical activity in reduction of CRP.[142324] Though in these researches, no indication was made to the effect of training type on the reduction of this biomarker. In general, one may conclude that, beside other treatments, Continuous and planned exercise may be used as an effective and complementary treatment to prevent and cure psychological disorders such as depression and anxiety, as well as cardiovascular diseases. Though, further research is needed to establish the effects of exercise types on reduction of psychological disorders and cardiovascular diseases.

TITLE: Comparing the Efficacy of Temperature-Controlled Radiofrequency Tonsil Ablation versus CO2-Laser Cryptolysis in the Treatment of Halitosis ABSTRACT.INTRODUCTION:: Halitosis and foreign body sensation are two common and disturbing symptoms of chronic caseous tonsillitis (CCT). The aim of this study was to compare the efficacy and safety of temperature-controlled radiofrequency (TC-RF) tonsil ablation with CO2-laser cryptolysis (CO2-LC) in the treatment of patients with halitosis caused by CCT. ABSTRACT.MATERIALS AND METHODS:: Sixty-two patients who suffered from halitosis and/or foreign body sensation due to CCT were enrolled in the present randomized clinical trial, and were randomly assigned into two groups. Group A underwent TC-RF tonsil ablation and Group B received CO2-LC. The severity of symptoms including halitosis and foreign body sensation was reported 7 days, 1 month, and 6 months after the procedure. Patient pain levels and amount of bleeding were evaluated as safety outcome measures. Pain levels were evaluated during the intervention, and at Day 1, 3, and 7 following the procedure using a visual analog scale (VAS). ABSTRACT.RESULTS:: Mean rank of pain score in the RF tonsil ablation group was found to be higher than in the CO2-LC group at all measured timepoints following the procedure. The amount of bleeding in the LC group was found to be significantly less than in the RF group (P<0.05). No significant difference was found between the groups regarding duration of procedure (P=0.157). ABSTRACT.CONCLUSION:: Both procedures were found to be effective and safe in the treatment of CT-associated halitosis. However, LC showed better results based on lower pain levels, lower incidence of bleeding, and faster progression to a routine diet. BODY.INTRODUCTION: Palatine tonsils contain tubular crypts which extend from the tonsillar surface through the deep layers of the parenchyma. These crypts may retain keratin debris, exfoliated epithelium cells, and foreign particles, and lead to an accumulation of a yellowish soft mass termed caseum. Therefore, palatine tonsils represent an appropriate medium for active anaerobic bacteria (1-3). Chronic caseous tonsillitis (CCT) is a common disease and has been frequently linked with halitosis (4). Indeed, it has been estimated that about 77% of patients with CCT complain of halitosis (5). Halitosis, an unpleasant odor present in the exhaled breath, can result in the social isolation of the patient and may eventually cause depression. Anaerobic proteolytic bacteria decompose organic materials and produce odoriferous compounds such as volatile sulfur, hydrogen sulfide, methyl mercaptans, and dimethylsulfide, which consequently leads to halitosis (5,6). CCT is characterized by caseum formation inside the tonsil crypts that may cause inflammation, leading to congestion and hypertrophy of the tonsils. Therefore, symptoms such as throat irritation and sensation of foreign bodies are common in CCT. CCT affects both men and women at any age, irrespective of their size, and can occur on one or both sides (1,2). The initial approach for the treatment of CCT includes the use of topical antiseptics, anti-inflammatory agents, and oral antibiotics (2,7). If these treatments do not improve symptoms, surgical excision of the tonsils is indicated. However, tonsillectomy is an invasive procedure with complications such as pain and bleeding that can potentially be more serious in adults (7). Thus, some conservative methods such as temperature-controlled radiofrequency (TC-RF) tonsil ablation and CO2-laser cryptolysis (CO2-LC) are recommended (7-15). This study was designed to compare the efficacy and safety of TC-RF and CO2-LC in the treatment of halitosis and alleviation of foreign body sensation in patients with CCT. To the best of our knowledge, the efficacy and safety of these procedures has not been evaluated in the treatment of halitosis to date. BODY.MATERIALS AND METHODS: This randomized clinical trial was conducted at Besat Hospital, affiliated to Hamadan University of Medical Sciences, from February 2013 to September 2014. Sixty-two patients suffering from halitosis and/or foreign body sensation due to CCT who were referred to the ear, nose and throat (ENT) clinic of Besat Hospital were enrolled in the present randomized clinical trial. Sample size selection was calculated according to previous studies of TC-RF tonsil ablation and LC for the treatment of halitosis (7,14). Inclusion criteria were as follows: minimum age of 18 years; positive Finklestein’s tonsil smell test; no response to routine medical treatment (250 mg metronidazole oral tablets three times a day for 10 days and gargling of normal saline for 1 month); and normal coagulation tests. Patients with the following criteria were excluded from the trial: periodontal diseases; gingivitis; sinusitis; nasopharyngeal, oropharyngeal, or laryngeal cancer; history of other systemic diseases that result in halitosis such as hepatorenal, pulmonary, gastroenterological diseases (such as gastroesophageal reflux), or diabetes mellitus. Moreover, patients who had contraindications to RF treatment of the tonsils (e.g. having a cardiac pace maker, immunodeficiency diseases, or coagulation disorders) and those who had limitations with regards to CO2-LC (e.g. anatomical oropharyngeal anomalies, Mallampati grading 3 or 4, cleft palate, limitations in terms of opening the mouth, and other oropharyngeal abnormalities) were excluded from the study. The present study was approved by the ethics committee of Hamadan University of Medical Sciences and all participating patients were informed of the study procedure, and signed written consent forms. Moreover, the study protocol was registered in the Iranian Registry of Clinical Trials(ID: IRCT201303233186N3). The origin of halitosis was established by Finkelstein’s tonsil smelling test which is based on massaging the tonsils and smelling the squeezed discharge. The test was performed by the examiner who pressed the anterior pillar with a tongue depressor in order to obtain caseum, and allowed the patient and his or her close family member to smell the resulting odor. If the fetid odor of caseum reflected the typical malodor of the patient, the test was considered positive. In patients who had foreign body sensation, if removal of the caseum led to improvement in the symptom, diagnosis was established. The severity of halitosis and foreign body sensation was evaluated preoperatively using a visual analog scale (VAS) such that a score of 0 represented no related symptoms while a score of 10 indicated the worst experience of related symptoms. Patients were randomly assigned to two groups using balanced block randomization. For this purpose, four sheets of paper were prepared. The letter “R” meaning “radiofrequency” was written on two sheets and the letter “L” meaning “Laser” was written on the other two sheets. The paper sheets were then placed in a container. An independent third party randomly took one paper at a time from the container for each patient without replacing the paper until the four sheets were removed. After that, all paper sheets were placed back into the container and the action was repeated several times to reach the required sample size. Surgical technique Both procedures were performed under local anesthesia with lidocaine spray and an injection of lidocaine 2% at the anterior and posterior pillar. All caseum from both tonsils was removed by applying pressure on tonsils. TC-RF tonsil ablation was achieved with the insertion of an RF probe into the tonsillar crypts at 10 W power (Celon lab ENT, Model: REF LAB 006.025.001) (200-970 V). CO2-LC was performed using an A.R.C LASER (GmbH model C-Las Type) with a nominal power of 30 W, attached to an articulated arm handpiece, with applications of 10 W continuous wave laser power, with a focus point of 2 mm. The handpiece did not touch the tonsils. Protective glasses were used by the patients and the medical team. According to the Brodsky scale, grade 1 and 2 tonsils received RF or laser beam in 4 points, and grade 3 and 4 tonsils received the same in 6 points (16). All patients were kept under observation in the hospital for 4 hours following the procedure, and were discharged with the recommendation to eat soft foods, gargle normal saline two times a day, and take 325 mg acetaminophen every 6 hours if required. Patient pain levels and amount of bleeding were evaluated as safety outcome measures. Pain level was evaluated during the intervention, and at Day 1, 3, and 7 following the procedure using a VAS. Patients were also asked after how many days following the procedure they were able to return to a regular diet and were able to go back to work. The amount of bleeding during the procedure was recorded using the following grading: Grade 0, no bleeding; Grade 1, some oozing requiring no intervention; Grade 2, minimal bleeding which could be controlled by compression; Grade 3, severe hemorrhage requiring surgical intervention. Patients were reexamined 7 days, 1 month, and 6 months following the procedure, and the severity of halitosis and foreign body sensation was recorded postoperatively using the following VAS: Score 0, no related symptoms; Score 10, worst experience of related symptoms. The amount of improvement was measured as follows: Improvement, >30% decrease in severity of symptoms; No improvement, <29% decrease in severity of symptoms. Data obtained were analyzed using SPSS software (version 16.0, SPSS Inc, Chicago, Illinois). Independent sample t-tests and the Mann-Whitney test were used to compare the efficacy of the procedures. Moreover, Chi-square tests were used for qualitative analyses if related. P-values less than 0.05 were assumed to be statistically significant. BODY.RESULTS: The study included 56 of the 62 patients initially enrolled. Twenty-eight patients underwent TC-RF tonsillar ablation (Group A) and 28 patients underwent tonsillar ablation with CO2-LC (Group B). Four patients were not available for follow-up (Fig.1). The proportion of male patients in Group A and Group B was 42.9% and 25%, respectively. The mean age ± standard deviation (SD) was found to be 27.8±8.1 years in Group A and 26.2±6.3 years in Group B. No significant differences were found between the two groups regarding age, gender, foreign body sensation, and halitosis before the study (P>0.05). Demographic characteristics of the patients are shown in (Table.1). Fig 1Enrollment and Follow-up Procedure of the Patients Table 1Demographic and Baseline Characteristic of two Groups Group A 1 (n=28) Group B 2 (n=24) P-Value Male 12 (43%) 6(25%) 0.44 Female 16 (57%) 18 (75%) Age mean (SD) 27.8 (8.1) 26.2 (6.3) 0.17 Foreign body sensation* 1.89 3.58 0.081 Halitosis * 5.35 3.54 0.100 *According to VAS mean range (between0-10) 1Group A: Temperature-Controlled Radiofrequency Tonsillar Ablation 2Group B: Co2- Laser Cryptolysis Degrees of improvement of halitosis and foreign body sensation in each group are shown in (Table 2,3), respectively. Seven days following the procedure, 68.4% of patients in the TC-RF group and 84.6% of the patients in CO2-LC group showed improvements in halitosis. Both procedures significantly improved the symptoms of halitosis and foreign body sensation (P<0.05). Moreover, significant differences were found between the groups in terms of improvement of halitosis at Month 1 (P=0.003) and Month 6 (P=0.014) following the procedure.Mean halitosis and foreign body sensation in the two groups before and at different time points after the intervention are shown in( Table.4). Mean rank of pain score of both groups (according to VAS) during intervention, at Day 1, 3, and 7 following the procedure are shown in (Table 5). Pain levels in Group A were greater than in Group B at all time points, and the differences between the two groups were statistically significant (P<0.05). Table 2:Degree of Halitosis Improvement at Different Time Points Following Interventions in Both groups Group A 1 (n=19) Group B 2 (n=13) p-Value Day 7                Improved, n (%) 13 (68.4) 11 (84.6) 0.192                Not improved, n (%) 6 (31.6) 2 (15.4) Month 1                Improved, n (%) 16 (84.2%) 10 (76.9%) 0.003                Not improved, n (%) 3 (15.8%) 3 (23.1%) Month 6                Improved, n (%) 15 (78.9%) 10 (76.9%) 0.014                Not improved 4 (21.1%) 3 (23.1%) 1Group A: Temperature-controlled radiofrequency tonsillar ablation 2Group B: CO2-laser cryptolysis Table 3Frequency of foreign body sensation at different time points following interventions in both groups Group A 1 (n=9) Group B 2 (n=11) p-Value Day 7                Improved, n (%) 7 (77.8) 9 (81.81) 0.028                Not improved, n (%) 2 (22.2) 2 (18.19) Month 1                Improved, n (%) 8 (88.9) 10 (90.9) 0.111                Not improved, n (%) 1 (11.1) 1 (9.1) Month 6                Improved, n (%) 7 (77.8) 10 (90.9) 0.028                Not improved, n (%) 2 (22.2) 1 (9.1) 1Group A: Temperature-controlled radiofrequency tonsillar ablation 2Group B: CO2-laser cryptolysis Table 4Mean of halitosis and foreign body sensation before and after the interventions in the two groups at different time points according to VAS Group A 1 Group B 2 Halitosis Foreign body Halitosis Foreign body Before, mean (SD) 5.35 (4.2) 5.88 (1.45) 3.54 (3.7) 7.16 (1.99) Day 7, mean (SD) 2.53 (3.2) 2.77 (2.48) 0.79 (1.8) 3.33 (3.02) Month 1, mean (SD) 2.57 (2.9) 2.44 (2.13) 1.04 (1.89) 3.5 (2.19) Month 6, mean (SD) 3.17 (3.69) 2.66 (2.12) 1.12 (1.94) 4.1 (3.01) 1Group A: Temperature-controlled radiofrequency tonsillar ablation 2Group B: CO2-laser cryptolysis Table 5Mean of halitosis and foreign body sensation before and after the interventions in two groups at different time points according to VAS During intervention Day 1 Day 3 Day 7 Group A1, mean 33.96 31.48 31.00 32.18 Group B2, mean 17.79 20.69 21.25 19.88 p-value, mean <0.001 0.010 0.018 0.001 1Group A: Temperature-controlled radiofrequency tonsillar ablation 2Group B: CO2-laser cryptolysis Six patients (21%) in the TC-RF group were reported to have oozing that stopped spontaneously. No bleeding was reported in the other patients. In the CO2-LC group, only one patient (4.1%) had oozing, which was significantly less than in the TC-RF group (P<0.05). There was no statistically significant difference between the groups regarding duration of procedure (P=0.157); however, the speed with which a regular diet was resumed was faster in Group B than in Group A. Patients had moved to a regular diet after 3.1 days in Group A and after 1.9 days in Group B (Table.6) Table 6Comparison of two groups regarding beginning of regular diet and duration of procedure Group A 1 (n=28) Group B 2 (n=24) p-Value Return to regular diet (day), mean (SD) 3.14 (2.32) 1.91 (1.05) 0.021 Duration of procedure (min), mean (SD) 6.41 (1.99) 6.10 (3.03) 0.157 1Group A: Temperature-controlled radiofrequency tonsillar ablation 2Group B: CO2-laser cryptolysis A statistically significant difference was found between the groups regarding the amount of time required to return to routine work following the procedure (P=0.052). No adverse effects or complications were encountered during the study. BODY.DISCUSSION: CCT is characterized by caseum retention and related symptoms such as halitosis and foreign body sensation. Failure of clinical treatment indicates tonsillectomy, which is likely to have side effects including, in particular, hemorrhage and pain. Recently, conservative procedures such as CO2-LC and TC-RF tonsillar ablation have been introduced. In these procedures, the crypt ostium is opened, thus avoiding caseum retention. In the present study, the efficacy and safety of the mentioned methods in the treatment of halitosis and alleviation of foreign body sensation in patients with CCT were compared. To the best of our knowledge, the efficacy and safety of these procedures has not previously been evaluation in the treatment of halitosis. According to the results of our study, halitosis and foreign body sensation were significantly reduced following both procedures. Pain level during the intervention and at all time points following the procedure was higher in the group who underwent TC-RF compared with CO2-LC. Dal Rio et al. previously evaluated the effects of CO2-LC in 38 patients with halitosis. According to their results, all patients showed improvement in halitosis after the procedure, which is consistent with the results of the present study. Moreover, volatile sulfur compounds were found to be decreased by 30.1%. Caseum retention was also significantly decreased (13). Finkelstein et al. studied the efficacy of CO2-LC in the treatment of halitosis in 53 patients with CCT. Complete elimination of halitosis was reported in 52.8% of the patients with a single session of CO2-LC. Two sessions were required in 34% of patients for complete elimination of halitosis, while three sessions were needed in 9% of patients. Overall, resolution of halitosis was reported in 92% of patients. In our study, 6 months following the procedure, patients who underwent CO2-LC showed improvement in halitosis and foreign body sensation by 76.9% and 90.9%, respectively. Consistent with results of the present study, Finkelstein et al. concluded that CO2-LC is an effective, safe, and well-tolerated procedure for the treatment of halitosis in patients with CCT (7). In a retrospective study, Tanyeri et al. evaluated the efficacy and safety of TC-RF tonsil ablation in the treatment of halitosis caused by CCT in 58 patients. complete elimination of halitosis was reported with one session in 84.4% patients and after two sessions in 6.9% patients,totally 91.3% had complete disappearance of halitosis. These findings support the results of our study. In our study, 6 months following the procedure, patients who underwent TC-RF tonsil ablation showed improvement of halitosis and foreign body sensation by 78.9% and 77.8%, respectively. Moreover, similar to the results of our study, the authors concluded that TC-RF tonsil ablation is effective in the treatment of halitosis caused by CCT (14). In another retrospective study, effectiveness of RF cryptolysis in the treatment of chronic CCT-related halitosis was investigated. According to the results, mean VAS score was significantly reduced from 6.82±1.45 at the beginning of the study to 0.88±2.5 after 12 months. Moreover, following one session of RF cryptolysis, 76.5% of patients were found to be negative in Finkelstein's test (8). In a prospective trial, use of a bipolar electrode to administer RF cryptolysis was compared with use of a monopolar electrode in the treatment of patients with halitosis. Better results were found with bipolar RF cryptolysis in comparison with the monopolar case. Thus, bipolar RF cryptolysis was recommended for the treatment of halitosis and tonsillitis (15). In our study, the level of pain was found to be greater in the RF group than in the LC group. This is probably due to the possible damage done to the neurovascular system and muscles in the tonsillar bed. In our study, 21% of patients in the TC-RF group were reported to have oozing that stopped spontaneously. No bleeding was reported in other patients. Among patients who underwent LC, only one (4.1%) had oozing, which was at a significantly lower rate than in the TC-RF group (P<0.05). In the study by Tanyeri et al., only one out of 58 patients experienced a self-limited bleeding during the RF procedure. Additionally, one patient experienced bleeding 24 hours following the procedure and underwent tonsillectomy. Our findings are consistent with the results of previous studies and confirm that there is a minimal risk of bleeding during and after both TC-RF tonsil ablation and CO2-LC. Indeed, this is a great advantage of these procedures over tonsillectomy in the treatment of CCT-associated halitosis. However, it should be noted that in patients with a strong gag reflex, the most feasible treatment is known to be tonsillectomy. The difference between the two groups regarding advancement to a regular diet was statistically significant (3.1 days for RF versus 1.9 days for LC). In the study by Finkelstein et al., patients returned to a regular diet 1 to 3 days following LC, which is similar to the results of the present study (7). According to Karadag et al., RF ablation results in changes in the microbiology of the tonsils. These researchers found a statistically significant reduction in bacterial number following RF administration in comparison with the control group (10). Therefore, one may conclude that RF ablation probably leads to a reduction of halitosis for a short time. We followed the patients for 6 months, and no significant difference was found in the level of halitosis at Week 1 and Month 1 after the procedure. These findings have also been demonstrated by previous studies in which patients were followed up over a longer time period. According to our results, both procedures were found to be effective in the treatment of CCT-associated halitosis in adult patients. However, LC was found to be a better choice based on a lower level of pain and bleeding and faster progression to a routine diet. It should be noted, however, that LC costs much more than RF ablation, and is not indicated in all patients. For instance, patients with a strong gag reflex or anatomical abnormalities of the mouth or throat, and patients who have a reduced mouth opening are not good candidates for LC and might be better treated with RF ablation. BODY.CONCLUSION: TC-RF tonsil ablation and CO2-LC were both found to be effective and safe procedures for the treatment of CCT-associated halitosis, while avoiding the post-procedure morbidities and discomfort of tonsillectomy. However, LC probably has advantages over RF ablation due to lower levels of post-operative pain and faster progression to a routine diet.

TITLE: NAT2 genotype guided regimen reduces isoniazid-induced liver injury and early treatment failure in the 6-month four-drug standard treatment of tuberculosis: A randomized controlled trial for pharmacogenetics-based therapy ABSTRACT.OBJECTIVE: This study is a pharmacogenetic clinical trial designed to clarify whether the N-acetyltransferase 2 gene (NAT2) genotype-guided dosing of isoniazid improves the tolerability and efficacy of the 6-month four-drug standard regimen for newly diagnosed pulmonary tuberculosis. ABSTRACT.METHODS: In a multicenter, parallel, randomized, and controlled trial with a PROBE design, patients were assigned to either conventional standard treatment (STD-treatment: approx. 5 mg/kg of isoniazid for all) or NAT2 genotype-guided treatment (PGx-treatment: approx. 7.5 mg/kg for patients homozygous for NAT2*4: rapid acetylators; 5 mg/kg, patients heterozygous for NAT2*4: intermediate acetylators; 2.5 mg/kg, patients without NAT2*4: slow acetylators). The primary outcome included incidences of 1) isoniazid-related liver injury (INH-DILI) during the first 8 weeks of therapy, and 2) early treatment failure as indicated by a persistent positive culture or no improvement in chest radiographs at the8th week. ABSTRACT.RESULTS: One hundred and seventy-two Japanese patients (slow acetylators, 9.3 %; rapid acetylators, 53.5 %) were enrolled in this trial. In the intention-to-treat (ITT) analysis, INH-DILI occurred in 78 % of the slow acetylators in the STD-treatment, while none of the slow acetylators in the PGx-treatment experienced either INH-DILI or early treatment failure. Among the rapid acetylators, early treatment failure was observed with a significantly lower incidence rate in the PGx-treatment than in the STD-treatment (15.0 % vs. 38 %). Thus, the NAT2 genotype-guided regimen resulted in much lower incidences of unfavorable events, INH-DILI or early treatment failure, than the conventional standard regimen. ABSTRACT.CONCLUSION: Our results clearly indicate a great potential of the NAT2 genotype-guided dosing stratification of isoniazid in chemotherapy for tuberculosis. BODY.INTRODUCTION: The annual incidence of tuberculosis is still over nine million cases worldwide [1]. With supervision by The Stop TB Strategy, the World Health Organization (WHO) has pursued standardized treatments [2]. The most effective treatment has been a standard 6-month four-drug regimen for the initial treatment of drug-susceptible pulmonary tuberculosis [3, 4]. However, several drug-related adverse reactions can result in discontinuation of the treatment [4, 5]. There can also be considerable morbidity, even mortality, particularly with drug-induced liver injury (DILI) [6–8]. These events incur substantial additional costs because of the increased frequency of outpatient visits, laboratory tests, and hospitalization in more serious instances. Alternative agents can have greater problems with toxicity and are often less effective, and treatment could be prolonged despite attendant challenges to ensure compliance. As a result, the risk of treatment failure and relapse would become even higher. Therefore, individualized drug management using four standard drugs to minimize the incidence of adverse drug reactions is an unmet need in the present clinical circumstances. Isoniazid (INH), a key drug of the currently recommended regimen for patients with tuberculosis, is metabolized by genetically polymorphic N-acetyltransferase 2 (NAT2) [9–11]. The rate of elimination of INH is trimodally distributed in accordance with NAT2 metabolic activity [12, 13]; NAT2 activity status is genetically controlled and basically depends on the number of active alleles (NAT2*4 and *12). Previous findings indicate that a genetic polymorphism is associated with large interindividual differences in the toxicity and efficacy of INH [13–19]. Slow acetylators, patients without any active alleles, develop hepatotoxicity more often than rapid acetylators, patients with two active alleles, during treatment for tuberculosis with the standard regimen [18, 19]. In contrast, rapid acetylators are prone to treatment failure with the standard regimen [13–16], probably due to insufficient exposure to INH. These observations imply that current dosage of INH recommended internationally is too much for slow acetylators and insufficient for rapid acetylators. Thus, we should assume that pharmacogenetically stratified treatment has the potential to avoid unfavorable outcomes with improvements in the cure rate. Before the start of a prospective clinical trial with the NAT2 genotype-guided regimen, dose-finding pharmacokinetic studies were independently carried out in Europe and Japan to expect appropriate clinical dosages of INH for each NAT2 genotype [20, 21]. Based on pharmacokinetic parameters estimated from these dose-finding studies, a genotype-based dosage for the present study was determined as follows; half the standard INH dose for slow acetylators, 1.5 times the standard dose for rapid acetylators, and the standard dose for intermediate acetylators. Here, we report outcomes of a randomized controlled trial for pharmacogenetics-based tuberculosis therapy (RT-PGTT) conducted in Japan (ClinicalTrials.gov Identifier: NCT 00298870). The purpose of this study was to elucidate whether stratified medicine based on the NAT2 gene polymorphism could improve the tolerability and efficacy of multidrug therapy for pulmonary tuberculosis with INH. BODY.METHODS: BODY.STUDY DESIGN: This study was designed as a multicenter, two-treatment, parallel group, prospective, randomized, open-label, blinded-endpoint (PROBE) controlled trial, comparing NAT2 genotype-guided and standard dosing of isoniazid in the intensive phase of the 6-month four-drug standard treatment for patients with newly diagnosed pulmonary tuberculosis. This study was approved by the Institutional Review Boards of Osaka University and the participating hospitals, and has been registered on ClinicalTrials.gov. The participating hospitals were National Hospital Organization Toneyama National Hospital, Osaka Prefectural Hospital Organization Osaka Prefectural Medical Center for Respiratory and Allergic Diseases, National Hospital Organization Kinki-Chuo Chest Medical Center, and Japan Anti-Tuberculosis Association Osaka Hospital, all in Osaka, Japan. This study was conducted according to the principles of the Declaration of Helsinki and in compliance with applicable regulatory requests. All of the patients being tested gave written informed consent before the study. BODY.STUDY POPULATION: The eligible population was identified from a series of patients newly diagnosed with pulmonary tuberculosis (men and women, 20–75 years old), requiring the 6-month four-drug standard treatment for the first time. The exclusion criteria in the pre-study screening phase were as follows: abnormal test results for liver and kidney function (serum aspartate aminotransferase; AST > 45 (IU/L), alanine aminotransferase; ALT > 50 (IU/L), alkaline phosphatise; ALP > 444 (IU/L), total bilirubin > 1.6 (mg/dL), and creatinine > 1.4 (mg/dL)) before the study treatments commenced, long-term use of steroids and/or immunosuppressants, inadequate clinical conditions such as hyperglycemia diabetes mellitus, acute life-threatening chronic progressive disease, pregnancy or lactation, and alcoholism. Patients not expected to complete the study protocol for social reasons were not recruited. After the allocation of the patients, Mycobacterium avium complex (MAC) infection and resistance to INH were examined and used as additional exclusion criteria to ensure the efficacy of INH. Patients with complex infections or INH resistance were excluded from the final analysis as illustrated in Fig. 1.Fig. 1Trial profile. Abbreviations: RA-type, rapid acetylator genotype homozygous for NAT2*4; IA-type, intermediate acetylator genotypes heterozygous for NAT2*4; SA-type, slow acetylator genotypes without NAT2*4; PGx, NAT2 genotype-guided treatment in which INH dosage was pharmacogenetically stratified based on individual NAT2 genotype; STD, conventional standard treatment with standardized dosage for all; Low-dose, half the conventional standard dose; High-dose, 1.5 times the standard dose; St-dose, conventional standard dose; INH-DILI, Drug-induced liver injury associated with isoniazid BODY.RANDOMIZATION AND STUDY MEDICATION: Before the allocation of a consenting patient, an individual NAT2 genotype status was determined at the central laboratory and office in parallel with clinical pre-study screening at each site. Qualified patients were assigned randomly to the pharmacogenetic-guided treatment (PGx-treatment) or the conventional standard treatment (STD-treatment) according to a computer-generated dynamic randomization schedule, which minimized the imbalance of patient backgrounds including NAT2 genotype status (rapid-, intermediate-, and slow-acetylator genotypes), study site, body weight, age, and baseline γ-glutamyltranspeptidase. Within 72 h after a blood sample was drawn for genotyping, site investigators were informed of only the dose of INH assigned to each patient (see Table 1). INH dosage for patients with more than 80 kg in body weight was not set up, in consideration for average body sizes of the Japanese population. The allocation and NAT2 genotype status of patients were semi-concealed from all clinical staff and patients.Table 1Dose of isoniazid in NAT2-genotype-guided treatment or conventional standard treatment NAT2 statusSA-typeIA-typeRA-typeBody weight (kg)<4040≤<4040≤<4040≤Dose of isoniazid (mg, once a day)   PGx-treatment 100 150 200 300 300 450   STD-treatment 200 300a 200 300a 200 300a Isoniazid tablet (100 mg, 50 mg) was used practically. Stratified dose was 2.5, 5, 7.5 mg/kg b. w. for patients with SA-, IA-, and RA-type, respectively. SA-type, slow acetylator genotypes without NAT2*4; IA-type, intermediate acetylator genotypes heterozygous for NAT2*4; RAtype, rapid acetylator genotype homozygous for NAT2*4. PGx-treatment, NAT2-genotype guided treatment; STD-treatment, conventional standard treatment. aMaximum dose regardless of body weight according to guidelines of ATS and JATA In this trial, all patients were treated according to the Japan Anti-Tuberculosis Association (JATA)’s guideline with the exception of INH dosage; a 6-month regimen comprising INH, rifampicin, pyrazinamide, and ethambutol/streptomycin for the first 2 months followed by rifampicin and INH for 4 months. This treatment schedule is in line with the current standard treatment recommended internationally for adult respiratory tuberculosis [4]. Table 1 summarizes the assigned dose of INH for each parallel group. All patients started taking the standard oral dose (approx. 5 mg/kg b.w., once daily) throughout the course so as to avoid delay in initial treatment, which was the same dosing for the STD-treatment. For patients in the PGx-treatment, dosages were adjusted based on individual NAT2 status within 3 days. Modified daily INH doses were approximately 7.5, 5 and 2.5 mg/kg for rapid, intermediate and slow acetylators, respectively. Approximately 5 mg/kg of INH, as same as the standard dose, was given to all of the intermediate acetylators throughout. Regarding the other drugs for the standard regimen, standard daily doses of rifampicin (10 mg/kg, max. 600 mg/body), pyrazinamide (25 mg/kg, 1,500 mg/body), ethambutol (15 (20) mg/kg, 750 (1,000) mg/body), and streptomycin (15 mg/kg, 750 mg/body) were recommended with the following dose ranges allowed at the discretion of the physician in charge of treatment: rifampicin, 8–12 mg/kg; pyrazinamide, 20–30 mg/kg; ethambutol, 15–20 mg/kg; streptomycin, 12–18 mg/kg. Administration of vitamin B6 was also allowed at the discretion of the physician. Primary outcomes were evaluated at the 8th week. The same treatments continued for 6 months in both groups if patients did not have any clinical need to change the ongoing treatment. BODY.STUDY PROCEDURES: The baseline clinical assessment consisted of chest radiography, demographics (age, sex, body weight, and body height), sputum examinations, and serum biochemistry tests. Three consecutive sputum samples were collected for detection of M. tuberculosis which was performed by microscopy, culture and polymerase chain reaction (PCR) assay, and for susceptibility testing of M. tuberculosis to INH, rifampicin, ethambutol and pyrazinamide. A blood sample was drawn to determine serum concentrations of ALT, AST, ALP, bilirubin, creatinine, γ-glutamyltranspeptidase, albumin, C-reactive protein, and glucose, a full blood count, and the erythrocyte sedimentation rate. Sputum and blood samples were collected from all the patients to analyze the items above at weeks 1, 2, 3, 4, 6, and 8 during the intensive phase, and then monthly during the continuation phase for 6 months. At each point, patients were asked about any adverse events that might have occurred since the last point. Chest radiographs were taken at the 8th week, and the 24th week or the end of treatment. BODY.OUTCOME MEASURES AND ASSESSMENTS: The primary outcome measures were the incidences of unfavorable events in two different treatment regimens in population who required dose modification based on the NAT2 gene polymorphism: (1) the incidences of drug–induced liver injury associated with INH (INH-DILI) that occurred within 8 weeks of the treatments, and (2) the incidences of early treatment failure at the 8th week. The secondary outcome measures were other adverse events observed during the 8-week study period. INH-DILI was assessed according to the diagnostic criteria of the Manual for Serious Side-Effects of Drug-induced Liver Injury from the Ministry of Health, Labor and Welfare of Japan [22–24]. In brief, hepatocellular injury was defined as a more than two-fold increase in the upper limit of the normal (ULN) concentration of ALT alone or a serum ALT ratio/ALP ratio greater than 5, where the ALT ratio = ALT value/ULN of ALT, and ALP ratio = ALP value/ULN of ALP. Cholestatic injury was defined as an increase above two-fold of the upper limit of the normal range of ALP or a serum ALT ratio/ALP ratio less than 2. Mixed injury was defined as a serum ALT ratio/ALP ratio of between 2 and 5. Causality assessments showed a relationship to the INH administration if the total score was more than grade 3, i.e. ‘possible’. Early treatment failure was defined as a combined surrogate endpoint: (1) a positive sputum culture at the 8th week examination, or (2) no sign of improvement in the chest radiograph at 8 weeks for patients with a negative sputum culture at baseline. Secondary outcome measure was defined by the other adverse events during the 8 weeks of the intensive phase of the anti-tuberculosis therapy. All outcomes were determined by the members of a separate central end point adjudication committee who used standard definitions and were unaware of treatment allocation and NAT2 genotypes. An independent Data and Safety Monitoring Committee could advise early termination of the trial for safety, scientific or ethical reasons. BODY.NAT2 GENOTYPING: Genomic DNA was extracted from peripheral whole blood of each subject using a QIA amp Blood Mini Kit (QIAGEN). Six single nucleotide polymorphisms in the NAT2 gene were analyzed: 190C to T (NAT2*19), 191 G to A (NAT2*14), 341 T to C (NAT2*5), 481C to T, 590 G to A (NAT2*6), and 857 G to A (NAT2*7). Detections were separately duplicated for quality control of genotyping. In brief, the three mutations 481C to T, 590 G to A and 857 G to A were detected by a PCR-RFLP (restriction fragment length polymorphism) method as described previously [17]. After PCR amplification using specific primers, the PCR products were digested with the restriction enzymes Kpn I, Taq I, and Bam HI to determine the nucleotide substitution at 481, 590, and 857, respectively. A mismatch PCR-RFLP procedure was performed to detect the 341 T to C, 190C to T and 191 G to A substitutions, as described previously [20]. The restriction enzymes Dde I and Msp I were used for the PCR products to determine the nucleotide substitutions at 341 and 190/191, respectively. Alleles without any defective forms were defined as functional allele, NAT2*4. BODY.SAMPLE SIZE CALCULATION: On the basis of data from our previous study [18, 25], we assumed that the incidence of INH-induced liver injury in slow acetylators would be 65 % in the control arm (STD-treatment; conventional standard dosing of INH) and 15 % in the test arm (PGx-treatment; NAT2 genotype-guided dosing of INH). We hypothesized that the occurrence of early therapeutic failure in rapid acetylators would be decreased, from 35 % in the STD-treatment to 15 % in the PGx-treatment [26]. To achieve a power of 0.8 with an alpha of 0.05, we aimed to enroll 22 slow acetylators and 156 evaluable rapid acetylators in the trial. According to our calculations, a maximum sample size of 360 patients would be needed to test this hypothesis, allowing for a 15 % dropout rate, with frequencies of rapid, intermediate and slow acetylators estimated at 0.50, 0.42 and 0.08, respectively. A group sequential analysis would be performed if 16 slow acetylators were assessable. The enrollment would be stopped when p values regarding INH-DILI in slow acetylators were less than O’Brien-Fleming type stopping boundary determined by Lan-DeMets approach. Then, the trial would be finalized early because of ethical considerations. BODY.STATISTICAL ANALYSIS: The analyses were based on the intention-to-treat approach, including all assessable randomized patients. Since the patients with intermediate genotypes receive the same dose of INH irrespective of the results of the randomization, their data were analyzed as a combined group. A two-tailed significance test was used. Categorical data were compared using the χ2 test or Fisher’s exact test as appropriate and Cochran-Mantel-Haenszel test was used for multivariate analyses. Continuous variables are presented as the mean and standard deviation (SD) or 25 %, 50 % and 75 % percentiles as applicable and were analyzed by ANOVA with posthoc and the Student-t test (for parametric variables) and the Mann-Whitney U test (for non-parametric variables). The Lan-DeMets alpha spending function was employed for major outcomes. Multivariable logistic analyses were performed to evaluate the relationship between the incidence of unfavorable events and the baseline factors/covariates. Analyses were performed using SPSS ver. 11.0 (SPSS Inc.) and R ver. 2.10.1 (The R Foundation for Statistical Computing). BODY.RESULTS: BODY.PATIENT ENROLLMENT AND DEMOGRAPHICS: In all, 172 patients were randomly assigned to one of the two study regimens, the NAT2 genotype-guided dosing treatment (PGx-treatment) and the conventional standard dosing treatment (STD-treatment), as summarized in Fig. 1. The patients’ backgrounds showed a balanced distribution (Table 2). Eighty three patients were assigned to the PGx-guided therapy and 89 to the empirical therapy. Among them, 17 patients were not eligible for analysis since they met exclusion criteria after their allocation; 12 with primary INH-resistant tuberculosis and five with MAC infections. The rest of the 155 patients were infected with organisms sensitive to all first-line anti-TB drugs. The distribution of NAT2 genotype frequencies was similar to that in our previous study [18].Table 2Baseline characteristics of all randomized participants valid for evaluation of isoniazid-related liver injury and other adverse events on intention-to-treat bases ParametersNAT2 genotype statusSA-typeIA-typeRA-typep valueTreatmentsSTDPGxPGx/STDPGxSTDPatients (n) 9 7 64 44 48 Sex (M/F, n) 4/5 4/3 44/20 25/19 35/13 Age (years)a 35.0 48.0 48.5 50.5 51.0 0.614 (35.0–43.0) (36.0–58.0) (34.0, 60.0) (33.0, 57.8) (30.0, 60.2) Weight (kg)a 55.0 59.5 54.1 50.0 57.5 0.058 (52.8–59.8) (47.9–62.8) (50.1–62.2) (45.9–55.2) (47.6–63.8) AST (IU/L)a 18 17 18 16 19 0.165 (15–22) (17–27) (15–24) (13–20) (15–22) ALT (IU/L)a 12 15 14 12 15 0.066 (10–19) (9–24) (10–21) (8–16) (11–21) ALP (IU/L)a 241 244 238 255 242 0.696 (214–305) (195–329) (184–285) (208–304) (205–288) Total-bilirubin (mg/dL)a 0.3 0.5 0.6 0.5 0.6 0.338 (0.3–0.6) (0.5–0.8) (0.4–0.7) (0.4–0.6) (0.4–0.7) Creatinine (mg/dL)a 0.6 0.6 0.7 0.7 0.7 0.462 (0.5–0.7) (0.5–0.8) (0.6–0.8) (0.5–0.8) (0.6–0.8) Anti-tuberculosis drugs (Dose, mg/kg/day)b  Isoniazidb 5.5 (0.8) 2.6 (0.3) 5.5 (0.9) 8.8 (1.3) 5.5 (1.0) NA  Rifampicinb 8.7 (1.4) 8.9 (1.7) 8.7 (1.4) 9.5 (1.4) 9.4 (1.5) 0.076  Pyrazinamideb 23.7 (3.0) 20.7 (4.3) 22.1 (5.9) 22.7 (2.7) 22.8 (4.6) 0.676  Ethambutolb 14.5 (2.0) 15.4 (3.2) 14.4 (3.5) 15.3 (4.1) 0.555 Concomitant drug (%)  Vitamin B6 44.4 50.0 40.6 43.2 43.8 Primary sputum culture (n) of complex infection or INH resistance  Negative 1 0 21 8 8  Positive-low (< +++) 7 6 26 22 26  Positive-high (> = +++) 1 1 9 9 10 aData are presented as the median (25 %, 75 %). bData are presented as means (SD). There was no statistical difference between PGx and STD in IA. Five groups were compared by ANOVA. PGx, NAT2 genotype-guided treatment group; STD, conventional standard treatment group; RA-type, rapid acetylator genotype homozygous for NAT2*4; IA-type, intermediate acetylator genotypes heterozygous for NAT2*4; SA-type, slow acetylator genotypes without NAT2*4. NA, not applied BODY.PRIMARY OUTCOMES: BODY.DRUG-INDUCED LIVER INJURY ASSOCIATED WITH ISONIAZID (INH-DILI): Seven of nine slow acetylators in the STD-treatment experienced INH-related liver injury (INH-DILI) within the first 4 weeks, while none of the seven slow acetylators in the PGx-treatment did during the first 8 weeks (attribute risk difference; 77.8 %, p = 0.003, Table 3a). Conversely, few rapid acetylators in either group had INH-DILI during the first 8 weeks; 4.2 % (2/48 patients) in the STD-treatment and 4.6 % (2/44 patients) in the PGx-treatment receiving high-dose INH, which were importantly equivalent to the incidence in intermediate acetylators (4.7 %, 3/64 patients). These outcomes were not different in a population valid for early treatment failure. Multivariable logistic analysis using the baseline patient characteristics revealed that the NAT2 slow acetylator genotype was associated with INH-DILI in the STD-treatment, but not in the PGx-treatment at all, suggesting standard dose of INH is too much for slow acetylators. There was not any other factors and confounders associated with INH-DILI.Table 3Primary end point results in slow and rapid acetylator genotypes NAT2 genotype statusGroups(n)Incidence (%)UnivariateMultivariate ep valuesRelative risk (95 % CI)p valuesPooled odds ratios (95 % CI)a) Isoniazid-related liver injury SA-type PGx (7) 0.0 0.003a – NAd STD (9) 77.8 RA-typec PGx (44) 4.5 1.000 1.091 (0.160–7.42) NAd STD (48) 4.2 reference b) Early treatment failure SA-type c PGx (7) 0.0 0.475 – – 0.595 – – STD (9) 22.2 RA-type PGx (40) 15.0 0.013b 0.379 (0.166–0.866) 0.015b 0.274 (0.097–0.776) STD (43) 39.5 reference reference c) Unfavorable event, i.e., combined endpoints of isoniazid-related liver injury or early treatment failure RA+SA-type PGx (47) 17.0 0.001a 0.354 (0.177–0.707) 0.002a 0.229 (0.091–0.574) STD (52) 48.1 reference reference PGx=NAT2 genotype-guided dosing group, STD=Conventional standard dosing group aless than O’Brien-Fleming type stopping boundaries of the Lan-DeMets alpha spending function bless than Pocock type stopping boundaries csecondary end point of each NAT2 genotype status dnot applied because of no confounder, and dCochrane–Mantel–Haenszel test stratified by high-excretion quantity level of tubercle bacilli of sputum culture at the screening test BODY.EARLY TREATMENT FAILURE: The therapeutic effects of NAT2 genotype-guided dosing on early treatment failure against drug-sensitive tuberculosis are shown in Table 3b). The NAT2 genotype-guided regimen successfully reduced the incidence of early treatment failure in rapid acetylators; 15.0 % (6/40 patients) in the PGx-treatment and 39.5 % (17/43 patients) in the STD-treatment (relative risk; 0.379 [95 % CI: 0.116, 0.866], p = 0.013). The difference was distinct on multivariate analysis stratified by high-excretion level of tubercle bacilli (Pooled odds ratio; 0.274 [95 % CI: 0.097, 0.776], p = 0.015). One rapid acetylator in the STD-treatment who developed INH-DILI failed to achieve any clinical improvement by the 8th week of treatment. Importantly, two of nine (22.2 %) slow acetylators with INH-DILI in the STD-treatment (5 mg/kg) did not show any clinical improvement. In these cases, their standard treatment was interrupted due to the INH-DILI and it was inevitable to withdraw INH for one of them. In contrast, all the slow acetylators in the PGx-treatment achieved 8 weeks of treatment successfully, even with a lower dose of isoniazid than in convention therapy. BODY.COMBINED UNFAVORABLE EVENTS: The incidence of combined unfavorable events, defined by early therapeutic failure or INH-related drug-induced liver injury, was significantly lower in the combined population of rapid and slow acetylators; 17.0 % in the PGx-treatment and 48.1 % in the STD-treatment (relative risk; 0.354 [95%CI: 0.177, 0.707], p = 0.001 (Table 3c)). Furthermore, the risk reduction was more conspicuous when the subjects were stratified by the level of primary sputum culture; pooled odds ratios [95%CI] were 0.229 [0.091, 0.548] in combined population of rapid and slow acetylators. Although the dose of INH for intermediate acetylators in the PGx-treatment was the same as that in the STD-treatment, overall clinical usefulness of the PGx-guided therapy was compared with the empirical therapy: the combined unfavorable events were 20.5 % (16/78 patients) in PGx-guided therapy and 42.9 % (33/77 patients) in the empirical therapy (relative risk; 0.479 [95%CI: 0.288, 0.795], p = 0.003) (Fig. 2).Fig. 2Overall incidence of combined unfavorable events in the patients who received PGx-therapy and in those who received empirical therapy BODY.SUB-ANALYSES: The cumulative incidence of INH-DILI over time is illustrated in Fig. 3. Mean (SD) time to detect INH-DILI was 15.6 (6.9) days in slow acetylators receiving the conventional standard treatment. As for intermediate acetylators, INH-DILI, early therapeutic failure and the combined unfavorable events occurred in 4.7 % (3/64 patients), 26.8 % (15/56 patients) and 28.6 % (16/56 patients), respectively. Overall probabilities of the combined unfavorable events were 23.3 % (24/103 patients) in the PGx-treatment (8/47 patients) plus intermediate acetylators (16/56 patients) and 48.1 % (25/52 patients) in the STD-treatment.Fig. 3Cumulative incidence curves of isoniazid related drug-induced liver injury (INH-DILI) over time among the 172 patients. RA-type, rapid acetylator genotype; IA-type, intermediate acetylator genotypes; SA-type, slow acetylator genotypes; PGx, PGx-guided treatment; STD, conventional standard-treatment Further explorative analyses were performed in 118 patients presenting as sputum culture-positive at screening, who were infected with M. tuberculosis sensitive to first-line anti-tuberculosis drugs. As well as primary outcomes, PGx-guided treatment reduced INH-DILI with keeping enough therapeutic efficacies in slow acetylators, and also reduced incidence of persistent positive culture without increase of INH-DILI frequency in rapid acetylators (Fig. 4). Regarding persistent positive sputum culture at the 8th week, the risk difference was striking in rapid acetylators with highly-positive level of organisms on primary sputum culture; 10.0 % (1/10 patients) in the PGx-treatment and 66.7 % (6/9 patients) in the STD-treatment (p = 0.020). Incidence of unfavorable events of the combined population of rapid and slow acetylators was obviously higher in the STD-treatment than in PGx-treatment. In contrast, the PGx-guided dose stratification reduced unfavorable events to the level of intermediate acetylators’ (Fig. 5).Fig. 4Incidence of isoniazid induced liver injury (INH-DILI) and persistent positive culture among the patients with drug sensitive tuberculosis on sputum culture at screening. Closed columns, INH-DILI; open columns, persistent positive culture; RA-type, rapid acetylator genotype; IA-type, intermediate acetylator genotypes; SA-type, slow acetylator genotypes; PGx, PGx-guided treatment; STD, conventional standard-treatment Fig. 5Incidence of combined unfavorable events among the patients with drug sensitive tuberculosis on sputum culture at screening. RA + IA-type, combined with rapid or slow acetylator genotypes; IA-type, intermediate acetylator genotypes; PGx, PGx-guided treatment; STD, conventional standard-treatment BODY.SECONDARY OUTCOMES: Other adverse events observed during the 8-week study period were skin and subcutaneous tissue disorders including rashes (n = 48), nervous system disorders including headaches (n = 22), gastrointestinal disorders (n = 22), peripheral neuropathy (n = 13), visual impediment (n = 3), and hearing impairment (n = 3). Peripheral neuropathy was observed in two of nine slow acetylators in the STD-treatment, but not at all in the PGx-treatment (22 % vs 0 %). As for the other genotypes, peripheral neuropathy was observed in four of 48 patients who received the STD-treatment and three of 44 patients who received the PGx-treatment (8 % vs 7 %) in the group of rapid acetylators, and four of 64 (6 %) in the group of intermediate acetylators. Among the 13 patients presenting with this event, seven patients received vitamin B6: one slow acetylator and three rapid acetylators in the STD-treatment, one rapid acetylator in the PGx-treatment, and three intermediate acetylators. There was no statistically significant difference in the incidence of these adverse events between the PGx-treatment group and STD-treatment group. BODY.DISCUSSION: In the present study we have demonstrated the significant therapeutic potential of the NAT2 genotype-guided dosing of INH in the intensive phase of the internationally standardized 6-month four-drug regimen for newly diagnosed pulmonary tuberculosis. The new dosing regimen significantly reduced the incidences of INH-DILI and early treatment failure, compared to the conventional standardized regimen. The slow acetylators assigned to the PGx-treatment, who were administered a lower dose of INH than the STD-treatment, developed no INH-related liver injury, but achieved clinical improvements within 8 weeks. On the other hand, the rapid acetylators in the PGx-treatment, receiving a higher dose of INH, achieved a 25 % higher rate of clinical improvement by the 8th week than those in the STD-treatment, with an incidence of INH-related liver injury of less than 5 %. The absolute risk reduction in the combined population of rapid and slow acetylators was 31 % with respect to unfavorable events, i.e., INH-DILI or early treatment failure. NAT2 genotypic status has been focused on as a genomic biomarker for individualized medicine to preempt INH-related unfavorable events. In fact, INH-induced hepatotoxicity has been studied enthusiastically worldwide since we reported its association with slow acetylator genotypes [18]. In accordance with previous observational studies [18, 19], the present randomized clinical trial has confirmed that slow acetylators are prone to INH-DILI by conventional standard treatment with the relative risk of 4.5 (95 % CI, 1.3–15.3). Importantly, the present trial has clearly demonstrated that the incidence of INH-DILI was strikingly decreased by the NAT2-guided dosing stratification of INH. These results suggest that NAT2-guided dosing for slow acetylators would contribute to the preemptive discontinuation of anti-tuberculous drugs, thereby reducing the incidence of treatment failure and/or relapse of tuberculosis. It has also been discussed that rapid acetylators are prone to treatment failure during anti-tuberculosis therapy based on INH [13, 15, 16]. Consistent with this, we showed that the risk of treatment failure was higher in genotypic rapid acetylators, treated with the conventional dosage of INH, in the present study. Accumulating evidence has revealed the mechanisms of treatment failure with the four-drug standard regimen. Patients with low INH plasma concentrations have a propensity for treatment failure or early relapse of tuberculosis [27–29]. Indeed, in vitro and in vivo animal studies have demonstrated that the cumulative antibacterial effect of INH is associated with the area under the INH concentration versus time curve [30]. In combination therapy for tuberculosis, the main population of M. tuberculosis is susceptible to INH, while pyrazinamide is effective against slower growing bacilli in acidic milieus, and rifampicin against nonreplicating bacilli. During the first 2 to 5 days of the multidrug therapy, the bactericidal activity is derived mainly from INH [31, 32]. This bactericidal activity ceases due to the emergence of an INH-resistant population [33], resulting in treatment failure. In the present study, NAT2 genotype-guided dosing stratification of INH obviously improves therapeutic efficacy against INH-sensitive tuberculosis during the first 8 weeks, although the PGx-treatment could not start until a couple of days after the first dose. This fact strongly indicates that appropriate dose adjustment of INH at the beginning of therapy is important to avoid treatment failure and the relapse of tuberculosis, especially for patients infected with M. tubeculusosis sensitive to INH. Of note, the success rate for anti-tuberculosis treatment has been lower in Japan than in Western countries [1, 34]. Although a Japanese authority, JATA, has been propelling WHO-recommended programs, and has changed the standard INH dose from 400–450 mg to 300 mg, the treatment–success rate has not been improved in Japan. Global Health Observatory Data reported that smear-positive tuberculosis treatment-success rates were 52 % in Japan and 69–95 % in Europe in 2009 [1]. The lower success rate in the Japanese population might be explained by an interethnic difference in NAT2 genotype frequencies. The frequency of rapid acetylator genotypes in the Japanese population (approx. 50 %) is much higher than that in Caucasians (approx. 5 %) [12, 18, 35, 36]. Taken together, the dose of INH used in conventional treatment is insufficient for more than a third of Japanese with NAT2*4/*4 in achieving a satisfactory treatment-success rate. NAT2 genotype-guided dosing stratification would improve early treatment success rates against tuberculosis, especially in highly-positive excretion levels of bacilli, if the high dose of INH was applied from the first prescription exclusively to rapid acetylators. In conclusion, the present pharmacogenetics clinical trial with a PROBE-design demonstrated that NAT2 genotype-guided dosing stratification of INH could improve treatment outcomes in patients with drug-sensitive tuberculosis. Half of the conventional standard dose for slow acetylators reduced INH-DILI and did not reduce therapeutic efficacy. For rapid acetylators, 1.5 times the conventional standard dose of INH reduced early treatment failures without an increase in the incidence of INH-DILI. We propose here that the pharmacotherapy against tuberculosis with INH should be individually designed by stratification based on NAT2 genetic information in order to achieve the greatest success outcome for each patient.

TITLE: Effect of Dairy and Nondairy Calcium on Fecal Fat Excretion in Lactose Digester and Maldigester Obese Adults ABSTRACT.BACKGROUND: The effect of dietary calcium on fecal fat excretion in lactose maldigestion is not known. ABSTRACT.OBJECTIVE: To investigate the effect of dairy and nondairy dietary calcium on fecal fat excretion in lactose digesters and maldigesters during moderate energy restriction. ABSTRACT.DESIGN: A randomized cross-over trial comparing the effect of 500 mg vs. 1500 mg dairy and nondairy calcium on fecal fat excretion in 34 healthy adults during moderate (− 30%) energy restriction diet-induced weight loss for 12 weeks. The participants were classified as lactose digester or maldigester on the basis of breath hydrogen test. ABSTRACT.MEASUREMENTS: Anthropometric parameters and body composition, resting energy expenditure, energy and nutrient intake, fecal fat, physical activity, blood pressure, blood and urine sampling for pertinent measurements. ABSTRACT.RESULTS: Fecal fat loss expressed as percent of fat intake was significantly higher with 1500 mg (high-Ca) compared to 500 mg (low-Ca) calcium intake per day (mean: 3.0%; the 95% CI: 2.3 to 3.7%; P <0.001) independent of calcium source and lactose digestion status. ABSTRACT.CONCLUSIONS: During moderate energy restriction induced weight loss a high-Ca diet causes an increase in fecal fat excretion independent of calcium source. Calcium intake related fecal fat loss is also independent of the ability to digest lactose and it is not diminished over time. BODY.INTRODUCTION: There is an ongoing controversy over the role of calcium intake on body energy regulation. Low levels of dietary calcium and dairy products have been identified as a potential contributing factor to obesity and have also been linked to increased risk of hypertension and insulin resistance1–3. Despite supportive epidemiological reports2, 4–7, interventional and mechanistic studies8–12, the suggested anti-obesity effect of dairy and nondairy calcium supplementation remains far from proven because, not all investigations have confirmed these findings. Several studies indicate that calcium supplementation or dairy products may have no effect13–19 or even an adverse effect20 on body weight. Further, recent reviews and meta-analyses18, 21 of randomized controlled trials with or without concomitant energy restriction indicate that neither calcium supplementation nor dairy products reliably facilitate weight loss. When found, such a relationship relates to decreased rate of weight22 or fat gain, rather than weight or fat loss. Two recent trials23, 24 have reported over two-fold increase in fecal fat loss with increase in dietary calcium intake for 7 days. The authors concluded that their observation might contribute to an explanation why a high-calcium diet might be inversely related to the body weight. However, the long-term effects of calcium supplementation on fecal fat excretion are unknown. Moreover, there is no clear evidence that dairy- derived calcium would be more effective in causing fecal energy loss than nondairy calcium. Lactose maldigestion has been linked to low dairy calcium intake and osteoporosis25, although a preponderance of evidence suggests that lactose maldigestion should not limit calcium intake since most of the maldigesters can tolerate several servings of dairy foods daily26. However, the effect of lactose maldigestion on fecal fat loss secondary to dairy and nondairy calcium supplementation has not been systematically studied. The goal of the present study was to determine the effect of dietary calcium on fecal fat loss. We examined the association between lactose maldigestion, dietary calcium level and source, and weight loss induced by energy restriction and fecal fat loss for 12 weeks. We tested the hypothesis that dietary dairy and nondairy calcium affects energy balance by increasing fecal loss of fat. We also examined a possibility that fecal fat excretion might be different in lactose maldigesters than in lactose digesters. BODY.SUBJECTS AND METHODS: BODY.PARTICIPANT SELECTION: We studied healthy males (10) and females (24), aged 21–50 with BMI 29–35 kg/m2. Volunteers were recruited using flyers, massive emails, and word of mouth. Potential participants with history of medical illness including diabetes, hypertension, renal, liver, or heart disease, pregnant or lactating mothers, those taking medications or dietary supplements that affect body weight, lipid-lowering medications or thyroid hormone substitution, and engaged regularly in heavy or vigorous physical activities were excluded from the study. Volunteers with known or suspected drug or alcohol abuse and tobacco users were also not recruited. We certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research. The trial was conducted at the Clinical Research Center (CRC) at Vanderbilt University, Nashville, Tennessee, United States, was approved by the Institutional Review Board of Vanderbilt University, and followed the ethical principles of the Helsinki-II Declaration. Each participant provided written consent before the study. BODY.STUDY DESIGN: Based on breath hydrogen test, lactose digesters and maldigesters were randomly assigned in a single-masked manner to oneof two energy restriction diets: 1) dairy calcium in the form of dairy products and, 2) nondairy calcium in the form of food products excluding dairy. Each diet had two levels of dietary calcium; 500 mg (low-Ca) and 1,500 mg (high-Ca). Nondairy, high-Ca diet was supplemented with calcium citrate (1,000 mg of Ca). A sequence of the two levels of dietary calcium, either low-high calcium (500 – 1,500 mg) or high-low calcium (1,500 – 500 mg) was randomly assigned to each participant for 12 weeks (6 weeks each diet) in cross-over design. Adherence to the protocol was monitored by collecting self-reports of uneaten foods. Urinary nitrogen, sodium, and potassium were measured weekly in 24-h urine as biomarkers of intake. BODY.DIETS: Study participants received individualized energy and nutrient controlled diet for the whole study period provided by the CRC metabolic kitchen for consumption at home. The food items in these diets were similar to those regularly used in the U.S. and were adjusted to individual preferences. Energy needs were calculatedas sum of resting energy expenditure (REE) measured using metabolic cart (CPX Optima, MedGraphics, St. Paul, MN), energy expenditure of physical activity measured using accelerometer (RT3, StayHealthy, Monrovia, CA) for one week before the study, and thermic effect of food estimated as 10% of REE. The individualized diet (10-day diet cycle) provided in daily portions contained approximately 70% (± 210 kJ) of daily energy requirements, 52 –54% of energy from carbohydrates, 26–29% of energy from fat, 17–21% of energy from protein, and 17–21 g of fiber. To assure sufficient micronutrient content of the diet, participants received a multivitamin supplement daily (Nature Made, Mission Hills, CA). After six weeks energy content of the diet was lowered (420 to 840 kJ/day) to reflect changes in body weight. No restrictions were posed on amounts of energy-free foodssuch as water, coffee (without sugar or milk), diet soft drinks, salt, and pepper. All food items were marked (breakfast, lunch, dinner, snack), and foods for each day were packed in thermo-insulated bags. The dinner meals were pre-prepared and only needed to be heated. Each participant received a written daily list of foods at every diet pick-up (4 times a week). Any uneaten foods and any additional foods eaten by the participants were reported on daily sheets collected at food pick up. The study dietitian met with each participant weekly to discuss the diet, resolve any barriers and concerns related to food or specimen collection, and to encourage compliance. Each participant was allowed one or two days (i.e., birthday, holiday) on which additional foods could be eaten. Intake data was analyzed for energy and nutrient content using NDS-R database (Nutrition Data System, St. Paul, MN). BODY.LACTOSE DIGESTION: Lactose digestion was assessedby measuring alveolar breath hydrogen exhalation at 30 minute intervals for three hours following a lactose oral challenge, asdescribed previously26. Participants were classified as lactase deficient (lactose maldigesters) on the basisof a rise in breath hydrogen concentration of greater than 0.90μmol/L (>20 ppm) after ingestion of 25 g of lactosein 250 mL of water. BODY.BODY COMPOSITION AND ANTHROPOMETRIC DATA: The National Health and Nutrition Examination Survey (NHANES) protocols27 were followed for all anthropometrical measurements. Body weight was measured at baseline and thrice weekly within 0.1 kg using a calibrated beam platform scale (Detecto-Medic, Detecto Scales, Inc, Northbrook, IL). Height was measured at baseline within 0.5 cm using a calibrated wall-mounted stadiometer (Perspective Enterprises, Portage, MI). Waist circumference was measured in standing position to the nearest 0.1 cm at the midaxillary high point of the iliac crest at minimal respiration at baseline and after 4, 8 and 12 weeks of energy restriction diet. The averages of the two readings were used for analysis. All measurements were performed bythe same investigator. Body composition was determined by DXA at baseline and after 12 weeks using narrow fan-beam technology (GE Lunar Prodigy™ Madison, WI). Fat mass (FM) and fat-free mass (FFM) were determined, and FFM was further divided into lean body mass (LBM) and bone mineral content (BMC). For quality assurance and equilibration, a calibration block was scanned each morning. A spine phantom was scanned on a weekly basis; the coefficient of variation (CV %) was 0.7%. Total body water was measured using the bioelectrical impedance (Quantum-II Desktop, RJL Systems, Clinton Township, MI). The measurement was done promptly after DXA measurement by the same investigator. Data were entered into the software program provided by the manufacturer (Cypress, RLJ Systems, Clinton Township, MI). BODY.RESTING ENERGY EXPENDITURE (REE): REE was measured at baseline and after 12 weeks and was defined as the average EE during a 30-min period of lying in a supine position after a 30-min rest following an overnight fast (>10 h) using metabolic cart (CPX Ultima MedGraphics, St Paul, MN). BODY.PHYSICAL ACTIVITY: Daily physical activity was assessed using an RT3 accelerometer (StayHealthy, Monrovia, CA, US). The participants were instructed to maintain their habitual physical activity throughout the study. They wore theactivity monitor on their right hip while awake for the duration of the study. The results were downloaded weekly and total and physical activity energy expenditure were calculated using energy calculated from the amount of movement measured by the monitor and REE measured at baseline. Physical activity levels (PAL) were calculated by dividing total energy expenditure by REE for each monitored day. BODY.BLOOD PRESSURE: Blood pressure was measured 3 times a week in the reclining position after 10 min rest with automatically inflating cuff (Dynamap, General Electric, Milwaukee, WI, USA). BODY.URINE ANALYSES: The complete 24-h urine samples were collected weekly. Urine volume and density was measured and a sample of 10 ml was frozen at −70°C until further analysis in the CRC Core laboratory. Urinary calcium, sodium, and potassium were measured using Vitros 250 Analyzer (Ortho-clinical Diagnostics, Rochester, NY, USA). Urinary nitrogen content was measured using nitrogen analyzer (Antek instrument nitrogen system 9000NS, Antek Instruments, Inc., Houston, TX, USA). The nitrogen excretion in the urine was used as a biological marker for protein intake by multiplying the content of nitrogen in the urine by the factor 7.7228. The sodium content of the urine was used as a biological marker of sodium intake29 and the potassium in the urine divided by 0.77 was used as a biological marker of potassium intake30. BODY.BLOOD ANALYSES: Venous blood samples were drawn on the morning after an overnight fast at baseline, at 6 weeks, and on the last day of the study (12 weeks). Basal metabolic panel including glucose and routine hematological indices (hemoglobin concentration, hematocrit, red and white blood cells count) were analyzed in the Vanderbilt University Hospital Laboratory using standard methodologies. Blood for determination of insulin, leptin, parathyroid hormone (PTH), and vitamin D was centrifuged at 2800 × g for 15 min at 4°C. Serum was extracted and the samples were stored at −70°C until later analyses at the Vanderbilt Diabetes Research Center Hormone Assays Laboratory. BODY.FECAL FAT ANALYSES: All feces excreted during one weekend day (24 h) were collected in plastic containers during all 12 weeks of the study. In addition, the subjects completed a questionnaire on daily defecation frequency during the whole study. The fecal samples were weighed and frozen at −20°C until sent to the analytical laboratory (Arup Laboratories, Salt Lake City, UT, USA) for fecal fat analysis using gravimetric method. BODY.STATISTICAL ANALYSIS: Data are presented as means, standard deviations (s.d.) or ranges. We considered the weekly measurements of fecal fat excretion calculated as percent of fat intake as well as their average over 12 weeks as the major outcomes. Since the fecal fat excretion was consistent over 6 weeks within each calcium intake level and diet source (dairy or nondairy), the average of the fecal fat excretion over 6 weeks was used as the primary outcome in the final analysis. Random-effects models were used with generalized least square estimators and Huber/White/sandwich estimator of variance. As main effects in the model, calcium intake level (500 and. 1500 mg/day), diet source (dairy and nondairy), lactose-digestion status (digester and maldigester), and gender (male and female) were included. Differences between the means for the body fat and weight loss, urinary biomarkers, energy expenditure, and physical activity were analyzed by paired t-test. All tests were two-tailed, and a P-value of < 0.05 was considered significant. Analyses were performed with STATA 9.2 (StataCorp, College Station, TX) and R (www.r-project.org). BODY.RESULTS: A total of 51 volunteers were recruited for the study, 40 were randomized, and 34 completed the study (19 Caucasians, 11 African Americans, and 4 declaring other ethnicities). One participant was dropped due to noncompliance, and five withdrew between week 1 and week 8. Baseline and end of the study characteristics of participants who completed the study are shown in Table 1. There was no difference in initial weight and body composition between lactose maldigesters and digesters and between dairy and nondairy diets (all P >0.05). BODY.DIETARY INTAKE AND COMPLIANCE WITH THE STUDY DIETS: The participants had an average energy intake of 7.5 ± 1.5 MJ (Table 2). The average intake of energy from carbohydrates, fat, and protein was 54.5 ± 3.0%, 28.9 ± 2.6%, and 18.4 ± 1.85%, respectively. There were no differences between macronutrient and fiber content between the diets (dairy vs. nondairy). Average amount of calcium in low-Ca and high-Ca diets was 503 ± 73 and 1491 ± 131 mg/day, respectively and the level was not different between dairy and nondairy diets. There were no significant differences between the intake of protein, sodium, and potassium and their respective biological markers in weekly urine collections. Ratio of nitrogen in urine and corresponding protein intake were not significantly different between the dairy and nondairy diets (1.13 ± 0.41 and 1.01 ± 0.14, P = 0.105). There were also no differences between the ratios of reported intake and excretion between dairy and nondairy diets for sodium (0.96 ± 0.20 and 0.95 ± 0.19, P = 0.917) and potassium (1.15 ± 0.23 and 1.12 ± 0.16, P = 0.684). BODY.FECAL FAT EXCRETION: The average fecal fat loss (Table 3) over 6 weeks was significantly higher with high-Ca diet than that with low-Ca diet after adjusting for gender, lactose digestion status, and dairy or nondairy diet source (mean: 3.0%; the 95% confidence interval (CI): 2.3 to 3.7%; P < 0.001). The fecal fat loss increased correspondingly with cross-over to high-Ca diet and vice versa (Figure 1). It was also significantly higher in females than that in males after adjusting for calcium intake level, lactose digestion status, and dairy or nondairy diet source (mean: 2.0%; the 95% CI: 1.2 to 2.9%; P < 0.001). There was no significant difference in fecal fat loss between lactose digesters and maldigesters (Figure 2) after adjusting for gender, calcium intake level, and dairy or nondairy diet source (mean: 0.2%; the 95% CI: −0.5 to 1.0%; P = 0.544). There was a significant positive correlation between calcium intake and fecal fat excretion in dairy (r = 0.460) and nondairy (r = 0.555) diets and in lactose digesters (r = 0.562) and maldigesters (r = 0.463, all P >0.05). Although not statistically significant, the average fecal fat loss over 12 weeks was higher in nondairy than that in dairy diet group (Figure 1) after adjusting for calcium intake level, lactose digestion status and gender (mean: 0.5%; the 95% CI: −0.2 to 1.2%; P = 0.150). In females, the average fecal fat loss over 6 weeks tended to be higher with the non-dairy than that with the dairy diet after adjusting for calcium intake level and lactose digestion status (mean: 0.8%; the 95% CI: −0.1 to 1.6%; P = 0.067). BODY.URINARY CALCIUM EXCRETION: Urinary calcium excretion (Table 4) was significantly higher in high-Ca than low-Ca (128.3± 57.9 and 146.4 ± 77.9 mg/day−1; the 95% CI for difference: 3.8 to 31.1 mg/day; P = 0.014). The differences in calcium excretion were not different between dairy vs. nondairy diets and in digesters vs. maldigesters (all P > 0.05). BODY.BODY WEIGHT AND BODY COMPOSITION: After following an energy restricted diet for 12 weeks (Table 1), there were no significant differences in weight loss between dairy and nondairy diets (8.5 ± 2.7 and 7.2 ± 2.8 kg, P = 0.116). However, there were significant differences in weight changes in lactose digesters vs. maldigesters (9.2 ± 2.2 and 6.6 ± 2.4, P = 0.003). There was also significant differences between first and second 6-week study periods (5.6± 1.8 and 2.8 ± 1.7 kg, P= 0.001). The differences between dairy and nondairy diets were not significant during the first 6-week (5.7 ± 1.9 and 5.5 ± 1.7 kg) or second 6-week (2.9 ± 1.9 and 2.9 ± 1.8 kg) periods. There were also no significant differences in body fat changes across groups, dairy vs. nondairy (5.4 ± 1.5 and 4.1 ± 1.5; P = 0.393) or lactose digesters vs. maldigesters (5.5 ± 1.3 vs. 4.8 ± 1.7; P = 0.206). There were also no differences in the amount of total body water between the groups. BODY.RESTING ENERGY EXPENDITURE (REE) AND PHYSICAL ACTIVITY: There was a difference in REE between the baseline and the end of the study (7.31±1.31 vs. 6.74 ± 1.32 MJ) which became insignificant when REE was adjusted for fat free mass and fat mass (data not shown). There were not REE differences between digesters and maldigesters (Table 6). The differences in the amount of physical activity measured using RT3 accelerometers during the entire study were also not significant. Participants did not change physical activity level (PAL) during the study (Table 6). BODY.BLOOD PRESSURE AND BLOOD PARAMETERS: Diet had no significant effect on either systolic or diastolic blood pressure (Table 7). Basic hematological parameters were not different between groups (data is not shown). Diet did not have an effect on serum concentrations of insulin and vitamin D. Leptin levels decreased significantly after 12-week on the study diet independent of diet and lactose digestion status (40.6 ± 20.3 vs. 27.3 ± 16.7 ng/ml, P = 0.001, paired t-test). BODY.DISCUSSION: The novelty of the present study is that we investigated the random effect of dairy and non-dairy calcium intake on fecal fat excretion in lactose digesters and maldigesters during energy restriction-induced weight loss. The major finding is that high-Ca diet causes an increase in fecal fat loss independent of calcium source (dairy and nondairy) and ability to digest lactose. The effect of dietary calcium on fecal fat loss was not diminished over 12 weeks. The fecal fat loss increased correspondingly with cross over to high-Ca diet and vice versa as illustrated in Figure 1. Compared to low-Ca diet, high-Ca diet increased the fecal fat excretion by 1.8 g/day, or about 3 % of daily fat intake (6.7 ± 1.9 vs. 4.7 ± 1.8 g/day). This increase in fat excretion could produce 63 –125 kJ (15–30 kcal) per day of fecal energy loss, which could cause approximate 0.4 –0.7 kg body fat loss over the one-year period. This difference might be clinically relevant to long-term body weight regulation. The results of this 12-week investigation of effect of high calcium diet on fecal fat excretion are in line with prior short-term (1 to 2 weeks) human trials24, 31–33. For example, Denke et al31 have reported a 7% increase in fecal fat by increasing daily calcium intake from 410 mg to 2200 mg for 10 days. Shahkhalili et al32 observed an increase of 4 g/day in fecal fat by supplementing chocolate with 0.9 g calcium/day for 2 weeks. Welberg et al33 demonstrated a graded increase in fecal fat excretion (6.8%, 7.4% and 10.2%) with increasing calcium carbonate supplementation (0, 2 and 4 g per day) for a week. Very recently Bendsen et al23 and Jacobsen et al24 have reported an increase in fecal fat excretion of over 2 and 2.5 fold by increasing daily calcium intake to 2300mg and 1800 mg from 700mg and 500mg respectively. In a 7-day trial with dairy and non-dairy calcium supplementations, Boon et al34 also observed a non-significant trend towards a higher fat excretion on the high-calcium diet, noting a 56% higher fecal fat excretion on 2,500 mg vs. 400 mg calcium diet. Fecal fat loss in this study (8.2 g/day) was comparable or lower than fat loss reported in other studies. The differences may be largely explained by different calcium and protein contents of the diets used for the studies. It is known that a high protein intake increases calcium bioavailability leaving less calcium available for binding with fat in the intestine35, 36. For example, Jacobsen et al24 have noted modulation of the effect of high-calcium intake on fecal fat excretion by protein content of the energy intake. They have reported a fecal fat loss of 14.2 vs. 6.0 and 5.9 g/day with high calcium (1800 mg)-normal protein (15% of daily energy intake), low calcium (500 mg)-normal protein (15% of daily energy intake), and high calcium (1800 mg)-high protein (23% of daily energy intake) diets, respectively. The authors found no difference in fecal fat excretion between the low calcium-normal protein and the high calcium-high protein diets. We tested the effect of 1500 mg and 500 mg calcium diets with protein content of 17–21% of daily energy intake. We also observed that fecal fat loss was comparatively less stable in the first 6-week than second 6-week period (Figure 1). We do not have a definite explanation for that. Perhaps it might be related to greater weight loss in the first as compared to the last 6 weeks of energy restriction across all the variables studied. We cannot, however, compare our results to other studies since to the best of our knowledge no trial was reported with similar length (6 or 12 weeks) of controlled calcium intake and fecal fat excretion measurement. Our results further signify that fecal fat loss with a high calcium diet is not diminished over time, and this effect is not different in lactose digesters and maldigesters and with dairy and non-dairy calcium. In our study we did not observe any significant differential effect of dairy calcium on fecal fat loss. However, Lorenzen et al37 in a randomized crossover study with four isocaloric meals with differing amount or source of calcium reported a decrease in fat absorption with dairy calcium supplementation. It is not clear why calcium from dairy products but not calcium from supplement was found to inhibit fat absorption. The authors are of the opinion that dairy calcium is largely present as insoluble calciu23 have recently shown that increasing the daily intake of calcium from low-fat dairy products by 1600 mg for 7 days doubled the total fecal fat excretion (11.5 ± 1.4 g from 5.4 ± 0.5 g), but did not affect the excretion of bile acids. Their results suggest that fecal fat loss is not caused by binding of bile acids but rather it may be due to calcium soap formation in the intestine. Our results indicate that lactose maldigestion has no significant effect on fecal fat loss or urinary calcium excretion associated with dietary calcium. Approximately 70% of the world’s population loses the ability to digest large amounts of lactose after weaning, and this lactase nonpersistence is inherited as an autosomal recessive trait38. Several investigators have reported increased prevalence of osteoporosis in symptomatic lactose maldigesters39–41. Our findings are in line with the current understanding that inadequate calcium intake rather than lactose maldigestion per se is related to the consequences of calcium deficiency such as osteoporosis42,43. We noted that mean weight loss but not fat loss tend to be higher in lactose digesters as compared to lactose maldigesters. However, this study was not specifically designed to examine the effect of calcium on weight loss and nor does it have the power to support any such conclusions. All the participants were on isocaloric restriction and we found no significant differences in weight changes with the source of calcium after 12 weeks of energy restriction. Prior studies have reported conflicting effects of dairy or non-dairy calcium supplementation on weight loss, from no effect13–19 to significant effect8–12, 44. Our study must be interpreted within the context of our experimental design. The sample size was relatively small considering dietary calcium intake level, dairy or nondairy diet source, lactose digestion status, and gender as variables. However, it was a randomized controlled crossover trial with similar numbers of lactose digesters and maldigesters. Secondly, we did not measure fecal calcium but used urinary calcium as a marker of calcium intake. We also did not quantify fecal fat complexes (soaps) as a surrogate to soaponification. Rather, we relied on comparison of fecal fat excretion with low and high dietary calcium intake. Thirdly, 24-h stool samples were collected once per week. However, given constant content and reported intake of energy, macronutrients, and micronutrients including calcium of daily diets prepared by the metabolic kitchen it was reasonable to assume that stool fat content was also not different. Fourthly, the cross-over design was used only for dietary calcium level. Thus, the reason we could not detect the significant difference between non-dairy and dairy diet source could be due to large variability across subjects, constant amount of dietary calcium in respective diets, and possible gender differences. Our results for the effect of diet type or lactose digestion status would require further study. In summary, we found that during moderate (−30%) energy restriction-induced weight loss a high-Ca diet causes an increase in fecal fat excretion independent of calcium source. The new findings are that calcium intake related fecal fat loss is also independent of the ability to digest lactose and it is not diminished over time.

TITLE: Comparative study of anatomical landmark-guided versus ultrasound-guided suprascapular nerve block in chronic shoulder pain ABSTRACT.BACKGROUND:: Suprascapular nerve block (SSNB) is an effective method for the treatment of shoulder disorders. The present study was conducted to evaluate and compare the effectiveness of SSNB under ultrasonographic guidance with anatomical landmark-guided (LMG) technique in the treatment of chronic shoulder pain. ABSTRACT.MATERIALS AND METHODS:: A total of fifty patients with shoulder pain were enrolled in the present prospective randomized study. Patients in Group I (n = 25) received SSNB using the anatomical LMG as technique described by Dangoisse, in whom a total of 6 ml of drug (5 ml of 0.25% bupivacaine and 40 mg methylprednisolone) was injected. Group II patients (n = 25) were given SSNB using the ultrasound guidance with the same amount of drug. Pain was measured using visual analog scale (VAS), range of motion and Shoulder Pain and Disability Index (SPADI) were recorded. Observations were recorded before the block, immediately after the block, and 1 and 4 weeks after the block. ABSTRACT.RESULTS:: There was no statistically significant difference between the VAS score, range of motion and SPADI before the procedure (P > 0.05) in both the groups. Both the groups showed statistically similar improvement of VAS, range of motion and SPADI at 4-week (P > 0.05) follow-up. In Group I, VAS decreased from baseline value of 6.64 ± 1.50–2.04 ± 0.94 at 4 weeks (P < 0.001). In Group II, the VAS decreased from 6.92 ± 1.00 to 1.84 ± 1.03 at 4 weeks (P < 0.01). ABSTRACT.CONCLUSION:: In our study, both the techniques have produced comparable relief of pain, improvement in shoulder movement, and decreased SPADI 4 weeks after the block. BODY.INTRODUCTION: Chronic shoulder pain is a common complaint, especially among the elderly population leading to functional disability and decrease in quality of life. Shoulder pain may originate in the joint itself or from any of the surrounding muscles, ligaments, or tendons.[12] It has a prevalence of 15%–30% in the adult population. Activity modification, physiotherapy, and analgesics comprise the initial treatment in these patients. In several patients, it is difficult to treat as it responds poorly to conservative management (pharmacological and physical therapies) leading to progressive limitation of movement ultimately resulting in adhesive capsulitis. For this reason, it is important to consider interventional options such as suprascapular nerve block (SSNB) when conservative therapy fails.[123] A SSNB is an effective method for the treatment of shoulder disorders. It has been successfully used for the management of acute and chronic shoulder pain as well as for the diagnosis of suprascapular neuropathy.[4] SSNB has been found to be effective in common conditions that result in chronic shoulder pain which include rotator cuff lesions, adhesive capsulitis (frozen shoulder), calcifying tendinitis, shoulder arthritis, rheumatoid arthritis, and stroke sequel.[56789] The surface landmark technique of SSNB has undergone several modifications since it was first described by Wertheim and Rovenstien in 1941.[10] The most commonly used surface landmark technique of SSNB is the one described by Dangoisse et al.[11] The accuracy of surface landmark techniques can be improved using image guidance, which includes fluoroscopy, computed tomography (CT), and ultrasound. Ultrasound guidance has the advantage that it does not expose the patient and personnel to radiations, and it is a real-time procedure in which one can actually notice the infiltration of the drug around the suprascapular nerve (SSN) and recess site.[4] There are limited studies comparing efficacy of the SSNB using ultrasound-guided (USG) technique with the landmark-guided (LMG) technique in shoulder pain. Hence, we conducted this prospective study to compare and evaluate the anatomical LMG technique of SSNB with USG technique of SSNB in patients with chronic shoulder pain with regard to decrease in pain, increase in range of motion, and improvement of shoulder function. BODY.MATERIALS AND METHODS: This present prospective randomized study was conducted after the approval of Local Institutional Research Committee and written informed consent from the patient. Fifty patients of either sex, between 40 and 70 years of age, with chronic shoulder pain (visual analog scale [VAS] >4) of duration more than 3 months not responding to at least 2 weeks of oral analgesics and conservative therapy, referred to pain clinic were enrolled in the study. Patients with known contraindications for block interventions (e.g., infection at the site of block, coagulopathy etc.), history of adverse reactions to steroids and bupivacaine, uncontrolled diabetes mellitus, and patient not consenting for SSNB were excluded from the study. The patients were randomly divided in two groups of 25 each by a computer generated randomized number table. Patients in Group I (n = 25) were administered SSNB using the anatomical landmark technique described by Dangoisse et al.[11] Patients in Group II (n = 25) received SSNB using the USG technique. Both groups received same amount of drug. In Group I, nerve block was performed with patient in sitting position, after skin preparation and local anesthesia, a 21-gauge × 38 mm needle was introduced through the skin, 2 cm cephalad to the midpoint of the spine of scapula. The needle was advanced parallel to the blade of the scapula until bony contact was made with the floor of suprascapular fossa. After negative aspiration for blood, 5 ml of 0.25% bupivacaine and 40 mg methylprednisolone were slowly injected [Figure 1]. In Group II, patients received SSNB under ultrasound guidance using SonoSite M-Turbo ultrasound machine with 6–13 MHz linear probe. With the patient in sitting position and arm by the side, the ultrasound probe was placed in the coronal plane over the suprascapular fossa with slight anterior tilt. Suprascapular fossa was scanned from medial to lateral side to identify SSN and artery in the floor of fossa between the suprascapular notch and spinoglenoid notch. A 23-gauge Quincke spinal needle was used to pierce the skin after local infiltration with local anesthetic solution, in a mediolateral direction at an angle of 30–45 to the vertical, under the guidance of ultrasound [Figure 2]. After identification, 5 ml of 0.25% bupivacaine and 1 ml (40 mg) methylprednisolone were injected slowly under the visualization into the area around the nerve.[12] Figure 1The surface landmarks of Dangoisse's technique Figure 2Sonoanatomy of suprascapular region. Pointer showing the target site where the suprascapular nerve runs with the suprascapular artery covered by fascia of supraspinatus muscle The parameters to determine the efficacy of SSNB were pain, range of motion, and disability pain was assessed using VAS, 0–10 cm. Patients were familiarized with the use of VAS for the assessment of pain (where 0 denotes no pain and 10 is worst pain imaginable). Patients were asked to move the affected limb before rating their pain. Flexion, extension, abduction, internal rotation, and external rotation were recorded using a goniometer. Disability was assessed using Shoulder Pain and Disability Index (SPADI), which is a self-administered questionnaire that consists of two dimensions, one for pain and the other for functional activities. The pain dimension consists of five questions regarding the severity of an individual's pain, while dimensions for functional activities are assessed with eight questions designed to measure the degree of difficulty an individual has with various activities of daily living that require upper extremity use.[13] Pain and range of motion were recorded at the following intervals: before the procedure, 30 min, 1 week, and 4 weeks after the SSNB, while SPADI were recorded before the block, 1 week and 4 weeks after the block. Procedural complications and side effects such as pleural puncture, vascular puncture, hematoma at the injection site, rash, itching, numbness, tingling, and paresthesia if any were recorded. The primary end point of this study was assessment of SPADI score after 1 week. Based on earlier studies, we assumed that the difference of 10 in SPADI scores between the two groups was considered clinically significant, At two-sided type 1 error of 0.05, 90% power and standard deviation (SD) of 10, a sample size of 25 per group was required to detect a significant difference. Statistical testing was conducted with the statistical package for the social science system version SPSS 17.0 ((SPSS Inc. Released 2008. SPSS Statistics for Windows, Version !7.0 Chicago: SPSS Inc)). Continuous variables are presented as mean ± SD, and categorical variables are presented as absolute numbers and percentage. The comparison of normally distributed continuous variables between the groups was performed using Student's t-test and within the Group I (LMG) and Group II (USG), paired t-test was used. Nominal categorical data between the groups were compared using Chi-squared test or Fisher's exact test as appropriate. P < 0.05 was considered statistically significant. BODY.RESULTS: The data of all fifty patients were analyzed. The maximum number of patients in both the groups were the diagnosis of periarthritis shoulder followed by rotator cuff injury, postcerebrovascular accident sequel, impingement syndrome, and shoulder arthritis. The distribution of patients according to the age, sex, duration of pain, and diagnosis in the two groups was comparable (P > 0.05) [Table 1]. In Group I, the VAS for pain decreased from baseline value of 6.64 ± 1.50–2.12 ± 0.97 immediately after the block, further decreased to 2.12 ± 0.83 at 1 week and 2.04 ± 0.94 at 4 weeks. In Group II, the VAS decreased from baseline value of 6.92 ± 1.00–2.76 ± 1.30 immediately after the block, further decreased to 2.68 ± 1.25 at 1 week and 1.84 ± 1.03 at 4 weeks. Mean SPADI score improved from baseline score of 66.66 ± 10.79 in Group I and 65.07 ± 13.47 in Group II, to 34.24 ± 8.01 and 25.89 ± 14.30, respectively, at 1 week, and to 28.85 ± 5.19 and 24.37 ± 9.97 respectively at 4 weeks. The reduction of VAS in both the groups immediately after the block, after 1 week and after 4 weeks was found to be statistically significant (P < 0.001) in both the groups when compared with the baseline value. There was significant (P < 0.001) improvement in the SPADI at 1 week and 4 weeks after SSNB in both the groups [Table 2]. There was an overall significant (P < 0.05) improvement in all range of shoulder movements, i.e., flexion, extention, abduction, internal rotation, and external rotation in both the groups from the baseline value immediately following the SSNB which was maintained at 1 week and at 4 weeks after the procedure [Table 3]. Table 1Demographic data Table 2Comparison of visual analog scale and Shoulder Pain and Disability Index Table 3Comparison of range of motion There were no statistically significant differences between the two groups in VAS score, SPADI, and range of shoulder motion before the procedure (P > 0.05). The VAS score and range of motion of the two groups were statistically comparable with each other immediately after the block, 1 week and 4 weeks following the block. The USG group showed rapid improvement of shoulder function, had significantly (P = 0.02) better SPADI score than Group I at 1 week. The SPADI at 4 weeks in Group I and Group II was statistically insignificant (P = 0.054). Repeat block was required in 4 patients of Group I and 3 patients of Group II (P = 1.00.). Two patients in the Group I complained transient vagal symptoms which improved after some time and did not require any intervention. No complications were observed in Group II. The occurrence of complications in both the group was statistically comparable (P > 0.05). BODY.DISCUSSION: SSNB is a safe, simple, and effective technique for the management of chronic shoulder pain which can be easily performed in the outpatient department. In our study, both the techniques of the SSNB, i.e., USG and LMG resulted in decreased pain score, improved range of motion, and decreased SPADI scores after 4 weeks of administration of block. However, when both the techniques were compared with each other the improvement in pain score and shoulder movement and decrease in SPADI scores were comparable. None of them proved to be better than the other except USG technique resulted in early improvement in shoulder function (significantly lower SPADI scores) at 1 week. SSNB is being increasingly used for acute and chronic shoulder pain control and postoperative analgesia after shoulder surgery. SSNB is also preferred over other therapeutic options such as anti-inflammatory drugs and intraarticular steroid injections which have their limitations in the elderly population who have many comorbidities such as diabetes and renal dysfunction.[8141516] The SSN is mixed nerve, possessing both motor and sensory fibers, accounting for 70% of sensory supply to the shoulder joint, mainly the posterior and superior capsule. It originates from the ventral rami of the fifth and sixth cervical nerve roots. It emerges from the lateral aspect of the upper trunk of the brachial plexus, then courses posteriorly and laterally to the scapular notch. It enters the supraspinous fossa through the suprascapular notch below the superior transverse scapular ligament. The suprascapular artery and vein pass above this ligament. In the supraspinous fossa, the nerve is in direct contact with bone and exits the suprascapular fossa to infrascapular fossa lateral to the spinoglenoid notch. The superior articular branch given off in the supraspinous fossa, provides sensory supply to the the coracoclavicular, coracohumeral ligaments, the acromioclavicular joint, glenohumeral joint, and subacromial bursa. SSN also gives off motor branches for the supraspinatus and the infraspinatus muscle.[4171819] The SSN is targeted either in the suprascapular notch (posterior approach) or the supraspinous fossa (superior approach) to obtain interruption of sensory impulses; hence, it is important to know the anatomical details of the nerve. The posterior approach techniques guide the needle into the notch which may be absent in 15% of patients and may result in pneumothorax as the trajectory of the needle is toward the thoracic cavity. On the other hand, superior technique does not require the localization of nerve; the needle is directed in the lateral half of the fossa as supraspinatus muscle originates from the medial half. The advantages offered by this technique are extremely low risk of pneumothorax, ease of access, and it is not necessary to identify the notch.[4] The landmark guide technique used by Dangoisse et al. is a superior approach.[11] To improve the accuracy of the LMG techniques image guidance such fluoroscopy, CT and more recently ultrasound have been used. The technique of USG SSNB was first described by Harmon and Hearty in 2007. They suggested that the ultrasonographic view of the suprascapular region pertinent to the SSN block was the suprascapular notch and transverse ligament. The intended target of the ultrasound-guided injection was the notch.[20] Later, Peng et al. conducted a fluoroscopic and cadaveric study which suggested the reinterpretation of the sonoanatomy. When the US probe is positioned in the coronal plane over the suprascapular fossa with a slight anterior tilt, the SSN was visualized on the floor of the scapular spine between the scapular notch and the spinoglenoid notch. The concave shape of the floor was misinterpreted as the suprascapular notch, and the fascia of the supraspinatus muscle as the transverse scapular ligament. The target site for the SSN block was suprascapular fossa at this site where it forms a compartment, and the final needle-tip position was away from the notch with a potential decrease in the risks of pneumothorax or spread of local anesthetic toward the brachial plexus.[21] Hence, the superior approach LMG techniques and the USG technique target the nerve in the same area. Shanahan et al. conducted a study to compare anatomical landmark approach of SSNB versus CT-guided SSNB for shoulder pain in patients with degenerative joint rotator cuff disease. The patients were reviewed at 1, 4, and 12 weeks after injection. Similar to our findings, they observed that there were no significant differences in the improvement in pain and disability between the two approaches at any times. The study concluded that CT-guided and landmark approaches to performing SSNBs result in similar significant and prolonged pain and disability reductions and both approaches are safe.[22] Our results are also in consensus to study by Arcila Lotero et al. who evaluated the clinical efficacy and safety of ultrasound-guided SSNB in patients with chronic shoulder pain. They found significant improvement in VAS score after USG SSNB. In their study, the proportion of patients with reduced pain 2 days and 1 month after the procedure was 78.3% and 48.7%, respectively.[12] Ozkan et al. administered SSNB under fluoroscopic guidance using a nerve stimulator needle in patients with frozen shoulder and diabetes mellitus unresponsive to intraarticular steroid injections.[6] Gorthi et al. conducted a prospective randomized case–control study in fifty patients with perishoulder pain to analyze the effectiveness of SSNB under ultrasonographic guidance. Patients in the study group (n = 25) underwent nerve block using ultrasonographic guidance and control group (n = 25) patients were given the nerve block by without ultrasonographic guidance using a technique described by Moore. Degree of pain is assessed using a VAS and shoulder function was evaluated using the constant shoulder score (CSS). In contrast to our results, they observed that the study group showed better VAS and CSS patterns than the control groups at 1-month follow-up (P < 0.05). No complications occurred in the study group. In control group, there were two cases of arterial puncture and three cases of direct nerve injury with neurological deficit for 2 months.[23] The difference in results can be attributed to the Moore's technique of LMG SSNB used by them which targets the nerve in the suprascapular notch[24] while LMG technique used by us blocks the nerve in the supraspinous fossa. The probable reason for similar result in both the groups could have been due the volume used by us, i.e., 6 ml, which was sufficient to fill the lateral half of suprascapular fossa in all patients. Feigl et al. in a cadaver study concluded that 5 ml volume is enough to fill in lateral half of suprascapular fossa.[25] If we had used a smaller volume of drug, it could have produced different results, i.e., USG group could have been better because more targeted injections are given under guidance. USG SSNB is a new technique and there are only few studies which have compared LMG technique and USG technique of SSNB in chronic shoulder pain. The limitation of the present study was short-term follow-up of patients following the block; hence, long-term outcome of both the techniques could not be assessed. Another limitation was a small sample size, a large sample size could have helped us to validate our results more emphatically. BODY.CONCLUSION: The anatomical LMG described by Dangoisse et al. and the USG techniques of SSNBs are safe and efficient methods for the management of chronic shoulder pain. They decrease shoulder pain, increase range of movement, and improve shoulder functions. The two techniques used in the present study were comparable to each other in all aspects except the USG group resulted in early improvement of shoulder function. Since only a few studies have evaluated USG SSNB for management of chronic shoulder pain, further studies are required to evaluate and compare it with other LMG techniques of SSNB. BODY.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Effects of an Internet-Based Cognitive Behavioral Therapy (iCBT) Program in Manga Format on Improving Subthreshold Depressive Symptoms among Healthy Workers: A Randomized Controlled TrialInternet-Based CBT among Workers ABSTRACT.OBJECTIVE: The purpose of this study was to develop a new Internet-based computerized cognitive behavior therapy (iCBT) program in Manga format, the Japanese cartoon, for workers and to examine the effects of the iCBT program on improving subthreshold depression using a randomized controlled trial (RCT) design among workers employed in private companies in Japan. ABSTRACT.METHOD: All workers in a company (n = 290) and all workers in three departments (n = 1,500) at the headquarters of another large company were recruited by an invitation e-mail. Participants who fulfilled the inclusion criteria were randomly allocated to intervention or control groups (N = 381 for each group). A six-week, six-lesson iCBT program using Manga (Japanese comic) story was developed. The program included several CBT skills: self-monitoring, cognitive restructuring, assertiveness, problem solving, and relaxation. The intervention group studied the iCBT program at a frequency of one lesson per week. Depression (Beck Depression Inventory II; BDI-II) was assessed as a primary outcome at baseline, and three- and six-month follow-ups for both intervention and control groups were performed. ABSTRACT.RESULTS: The iCBT program showed a significant intervention effect on BDI-II (t = −1.99, p<0.05) with small effect sizes (Cohen's d: −0.16, 95% Confidence Interval: −0.32 to 0.00, at six-month follow-up). ABSTRACT.CONCLUSIONS: The present study first demonstrated that a computerized cognitive behavior therapy delivered via the Internet was effective in improving depression in the general working population. It seems critical to improve program involvement of participants in order to enhance the effect size of an iCBT program. ABSTRACT.TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000006210 BODY.INTRODUCTION: Occupational stress has been considered a major risk factor for a wide range of health outcomes [1], [2]. To reduce work-related stress and its negative effect on health among workers, more attention has been paid to the primary prevention strategy. Among other measures, stress management interventions (SMI) have recently become frequently applied in the workplace [3]. A growing body of literature has shown that SMIs based on cognitive behavioral therapy (CBT) [4], [5] and relaxation therapy [6] are effective in reducing work-related stress and improving depression and anxiety among workers. A meta-analysis reported that the estimated effects of CBT and relaxation therapy were Cohen's d = 0.68 and 0.35, respectively [7]. Another meta-analysis reported that the estimated effects of CBT and relaxation therapy were Cohen's d = 1.16 and 0.50, respectively [6]. However, the implementation of these interventions was still limited, because such a program requires professionals well trained in CBT [8]–[10]. In addition, even if a service is provided, the time, cost, and stigma related to mental health treatment could serve as barriers to access to the effective treatment [11]. An innovative way to deliver CBT-based treatment widely is with a computerized CBT (CCBT). CCBT programs can teach information and techniques based on the same CBT principles as face-to-face CBT programs do, with a highly structured format comprising educational lessons, homework assignments, and supplementary resources [12]. Spurgeon and Wright (2010) claimed that there were three major platforms on which to deliver the CCBT programs: 1) multimedia computer programs, 2) virtual reality programs, and 3) Smartphone or handheld device applications. Some programs are delivered in a clinic, and others are accessed at home or through other venues, such as the Internet [13]. The mode of application is an Internet-based cognitive behavioral therapy (iCBT): i.e., a CCBT delivered via the Internet. In addition to the features of CCBT, iCBT has a special feature of high anonymity and high accessibility. In terms of anonymity, iCBT is suitable for addressing psychological problems because it can enable users to avoid the stigma incurred by seeing a therapist [14]. In terms of accessibility, iCBT provides users with the opportunity to obtain treatment at any time and at any place, such as in the workplace or at home, and study the content as much as they want [15]. The level of therapist involvement in iCBT can vary among programs; they could provide no assistance, minimal therapist contact by email or telephone, or a contact similar to face-to-face therapy [15]. However, a complete online iCBT may have merit since it can provide a viable alternate mental health resource for people who have geographical, physical, psychological, and/or financial barriers to seeking traditional, face-to-face care [11]. Previous iCBT programs have been shown to have a significant positive treatment effect on depression and anxiety mostly in the clinical setting. A meta-analysis of RCT reported that iCBT can improve depressive symptoms of patients who have major depressive disorder (Hedges' g = 0.56–0.99) and anxiety disorders (Hedges' g values were 0.92 for social phobia, 0.83 for panic disorder, and 1.12 for generalized anxiety disorder) more effectively compared with TAU or waiting list [16]. The treatment effects of iCBT varied by the extent of therapist involvement. Another meta-analysis reported that the effect size (about 0.6 in Cohen's d on average) of iCBT with therapist support was greater than the effect size (about 0.25) of self-guided program without therapist support [15], [17]. However, these self-guided-only programs showed small to medium effects, and thus a self-guided iCBT program is still considered to be helpful for people who have mild to moderate symptoms but cannot (or do not wish to) see a clinician [12]. Most critical problem within self-guided programs that might have resulted in a smaller effect was high dropout rates [12]. Despite a number of benefits of using CCBT and iCBT, the application of these programs in the workplace setting has been limited. Grime (2004) conducted a randomized controlled trial (RCT) to evaluate the effect of an eight-week CCBT program to improve emotional distress among employees who had recently experienced stress-related absenteeism. A month before the end of the intervention, the depression and anxiety among the participants in the intervention group improved significantly compared to that of the TAU group [18]. Grime (2004) also reported reasons for non-participation [18]. Non-participation in this study was common and related to a number of barriers, such as access problems, preference for other treatments (especially face-to-face counseling), time commitment, skepticism about the intervention, and employer connection. An online or Internet-based CCBT program might be effective in reducing concerns about travel and time off work, confidentiality, and connection with the employer; thus it appears to improve workers' participation more than a CCBT program. However, no iCBT program is available that would target specifically the general working population, the effectiveness of which would be tested with an RCT. The purpose of this study was to develop a new six-week iCBT program for workers in Japan and to examine the effects of the iCBT program on improving the symptoms of depression at three- and six-month follow-ups using a randomized controlled trial design among workers employed in private companies in Japan. In the iCBT program, we applied a “Manga” (the Japanese cartoon or comic story) format, that is now frequently used to communicate medical information in Japan[19], expecting participants' greater commitment to and better understanding of the contents of the program with using this communicative medium[20], [21]. BODY.METHODS: BODY.TRIAL DESIGN: The study was a randomized controlled trial. The allocation ratio of the intervention group to the control group was 1 to 1. The study protocol was registered at the UMIN Clinical Trials Registry (UMIN-CTR) (ID = UMIN000006210). The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1, Protocol S1 and Protocol S2. BODY.PARTICIPANTS: All workers in company A (N = 290) and all workers who belonged to three selected departments (N = about 1,500) at the headquarter of company B (the total employee size, c.a. 11,000) were recruited by an invitation e-mail. Both companies developed information technology systems and related services as their products. Those who were interested in participating in the study were asked to go to a research website to obtain full explanation of the study's aim and procedure and to input their baseline information if they agreed to participate. The inclusion criteria stated that participants could 1) not be diagnosed with a major depressive disorder in the past month (using the web-version of the WHO-Composite International Diagnostic Interview version 3.0 (WHO-CIDI 3.0) [22], 2) not be diagnosed with lifetime bipolar disorder (WHO-CIDI 3.0), 3) not have taken 15 sick leave days or more in total due to their own health problems during the past three months, and 4) not have received medical treatment for mental health problems during the past month. BODY.INTERVENTION: Participants who were allocated to the intervention group studied the new iCBT program called the Internet CBT program; Useful mental health solutions series for business. The program was a six-week web-based training course to provide stress management skills. This program was structured into six lessons, with one lesson given per week. Learning one lesson required about 30 minutes, including homework. This program can be used anywhere the Internet is available. One of the unique features of the program was that the training was provided along with a Manga (Japanese comic) story of a psychologist and a client worker to facilitate the understanding of the participants (Figure 1). Several merits of using a comic story and comic characters in education have been acknowledged through research. First, these materials help motivate individuals, and high motivation is useful to keep participants in the program [23]. Second, they facilitate easy learning. A program with text combined with comic stories would be easier for a learner to understand compared to a text-only program [23], [24]. Third, comic stories foster learners' interest in the program [24]. These merits might be applicable to education in the workplace, because most Japanese people of working age are familiar with comics. 10.1371/journal.pone.0097167.g001Figure 1BODY.A SCREEN SHOT OF AN INTERNET-BASED COGNITIVE BEHAVIORAL THERAPY (ICBT) PROGRAM DEVELOPED IN “MANGA” (THE JAPANESE CARTOON OR COMIC STORY) FORMAT IN THIS STUDY.: In terms of the CBT components of the program, included were self-monitoring skills, cognitive restructuring skills, assertiveness, problem-solving skills, and relaxation skills. At the end of each lesson, participants were asked to submit homework to facilitate their understanding, although it was voluntary. Participants who submitted their homework received feedback from trained staff (clinical psychologists). The iCBT program included self-monitoring skills (in lesson 2), cognitive restructuring skills (in lessons 3 and 4), assertiveness (in lesson 5), problem-solving skills (in lesson 6), and relaxation skills (in lesson 4). In this study, a cognitive restructuring method was adopted as the primary outcome and main cognitive approach, and this has been shown to be effective in reducing depression [25]. Assertiveness and problem-solving training as well as relaxation skills were chosen as supplementary behavioral approaches to enhance the effect of the program. BODY.INTERVENTION GROUP: Participants completed six weekly lessons and homework within the iCBT program. They were allowed to complete the six lessons within 10 weeks after the baseline survey. Participants were reminded by e-mail to complete each lesson and/or to submit homework if they had not already done so. Reminders were sent from the research office to the participants every Monday. BODY.CONTROL GROUP: Participants in the control group received an e-mail message once a month titled “Useful information for stress management.” Each e-mail message consisted of about 500 words in Japanese and included stress management tips. The contents were as follows: 1) How to have a good sleep, 2) The effects of your diet and ingesting alcohol for stress, 3) How to have a good holiday, 4) Relaxing by listening to music and taking a bath, and 5) The effects of exercise on stress. Participants were also able to use an internal employee assistance program service. In addition, an e-learning program on stress and stress management was provided to all employees at company B. This e-learning program contained only a few descriptions of CB-based approaches. BODY.OUTCOMES MEASURES: BODY.BECK DEPRESSION INVENTORY II (BDI-II): The BDI-II is a 21-item self-report inventory that measures depressive symptoms such as sadness, pessimism, suicidal thoughts or wishes, tiredness or fatigue, loss of energy, and loss of pleasure, among others [26], [27]. Each item is scored on a scale ranging from 0 to 3, with a higher score indicating more serious depressive symptoms. BODY.KESSLER'S PSYCHOLOGICAL DISTRESS SCALE (K6): Psychological distress was measured with the Japanese version of the K6 scale [28], [29]. K6 consists of six items assessing the frequency with which respondents have experienced symptoms of psychological distress during the past 30 days. The response options range from 0 (none of the time) to 4 (all of the time). The internal reliability and validity found in previous studies are acceptable [28]. BODY.JAPANESE VERSION OF DYSFUNCTIONAL ATTITUDE SCALE 24 (DAS24-J): The 24-item Dysfunctional Attitude Scale (DAS-24) is a short version of the Dysfunctional Attitude Scale, which is a self-report inventory measuring depressogenic schemata. Each item is scored on a scale ranging from one (totally disagree) to seven (totally agree), with a higher score indicating a more dysfunctional attitude [30]. The Japanese version has been developed and tested, and its reliability and validity have been established [31]. BODY.IMPROVEMENT OF KNOWLEDGE AND SELF-EFFICACY: Respondents were asked to rate their improvement of knowledge and self-efficacy regarding five components of the iCBT program (Stress management, Cognitive restructuring, Assertive communication, Problem-solving, Relaxation training). Knowledge improvement was assessed by asking participants, “How much knowledge do you have about…” and self-efficacy improvement was assessed by asking respondents “How confident are you that you can do….” Both items were scored on 5-point scale ranging from 0 (none) to 4 (enough). BODY.DEMOGRAPHIC CHARACTERISTICS: Demographic data, such as age, gender, marital status, occupation, education, chronic disease, and overtime hours during the past month were also collected. BODY.SAMPLE SIZE CALCULATION: A systematic review of psychological treatments, mostly CBT for subthreshold depression, yielded a Cohen's d of 0.42 (95%CI: 0.23 to 0.60) at post-test [32]. On the other hand, it was reported that the effect of preventive interventions on the incidence of major depressive disorders was smaller in the universal setting than that in the selective or indicated setting [33]. In order to detect an effect size of 0.30 or greater at an alpha error rate of 0.05 and a beta error rate of 0.10, the estimated sample size was 235 participants per arm. With the anticipated dropout rate of 30%, the necessary sample size was 336 participants per arm. The statistical power analysis was conducted using the G*Power 3 program [34], [35]. BODY.RANDOMIZATION: Participants who fulfilled the inclusion criteria were randomly allocated to intervention or control groups (N = 381 for each group). Stratified permuted-block randomization was conducted. Participants were stratified into four strata according to two factors: K6 score (5 or greater or less than 5) at baseline survey and company (A or B) to which they belonged. A stratified permuted-block random table was generated by an independent biostatistician. Enrollment was conducted by a clinical research coordinator, and assignment was conducted by an independent research assistant. The stratified permuted-block random table was password-protected and blinded to the researcher. Only the research assistant had access to it during the work of random allocation. BODY.STATISTICAL ANALYSES: A mixed-model for repeated measures conditional growth model analyses were conducted using a group (intervention and control) * time (baseline, three-month, and six-month follow-up) interaction as an indicator of intervention effect. Intention-to-treat analysis (ITT) was conducted. The Linear Mixed Model in the PASW Statistics 18.0 was used. The Number Needed to Treat (NNT) to reduce depressive symptoms or psychological distress to achieve one improvement from subthreshold depression was calculated. Referencing the cutoff scores of BDI-II and K6 of previous studies [26], [36], the improvement was defined to change the scores from 14 or higher to 13 or less on the BDI-II or to change the scores from 5 or higher to 4 or less on the K6. Effect sizes and 95% Confidence Interval (95% CI) were calculated using Cohen's d among those who completed the questionnaire at baseline and at a follow-up. The values of 0.2, 0.5, and 0.8 are generally interpreted as being suggestive of small, medium, and large effects, respectively [37]. Subgroup analyses were conducted separately among respondents who had high (5 or higher score of K6) and low (4 or less score) scores at baseline, with the cutoff score for K6 selected based on a previous report, which suggested that 4/5 is the optimal cutoff point for K6 in terms of discriminating community residents and patients with mood and anxiety disorders [36]. BODY.ETHICS: The Research Ethics Review Board of Graduate School of Medicine, the University of Tokyo (No. 3083) approved the study procedures. We prepared a website which contained full explanation of the study. Before the baseline survey, participants were invited to read the explanation on the website, and asked to click an “ BODY.CHANGES TO THE PROTOCOL: One major change made to the protocol is the inclusion criteria. Originally it was planned that the participants who scored 5 or more for the K6 on the baseline survey would be allocated. Before the commencement of this study, this criterion was canceled due to the anticipation of a low participation rate. With this, sample size was recalculated. Another change made to the protocol is the method of statistical analysis. Originally the per-protocol analysis was planned, but the ITT analysis was conducted in the present study due to the expectation to be conservative in superiority trials. In addition, NNT was calculated due to the description of the difference between a treatment and a control in achieving a particular outcome. BODY.RESULTS: BODY.PARTICIPANT FLOWCHART: Participant flowchart is shown in Figure 2. Participants were recruited from 2 companies (N = 1790), and 850 (47.5%) of them completed a baseline survey. Out of those, 88 had to be excluded because 20 did not fulfill criterion #1 (not diagnosed as major depressive disorder in the past 1 month), and 2 did not fulfill criterion #2 (not diagnosed as lifetime bipolar disorder), which were assessed using the web-version of WHO-CIDI 3.0. An additional 35 did not fulfill criterion #3 (not having taken 15 or more sick leave days in total due to their own health problems during the past 3 months), and 42 did not fulfill criterion #4 (not having gone to the hospital during the past 1 month). Seven of them did not fulfill criteria #1 and #4, and four of them did not fulfill criteria #3 and #4. The remaining 762 participants were randomly allocated to an intervention or control group (N = 381 for each). 10.1371/journal.pone.0097167.g002Figure 2BODY.PARTICIPANT FLOWCHART.: At 3-month follow-up, 270 (70.9%) participants in the intervention group and 336 (88.2%) in the control group completed the follow-up survey. At the 6-month follow-up, 272 (71.4%) participants in the intervention group and 320 (84.0%) in the control group completed the follow-up survey. At each follow-up, the response rate of the control group was higher compared to that of the intervention group. Reasons for dropping out were not assessed in this study. BODY.RECRUITMENT: Recruitment and the baseline survey were conducted during the period from September to October 2011. The intervention and control groups were assessed at approximately three months (from December 2011 to January 2012) and approximately six months (from March to April 2012) after the baseline survey. BODY.BASELINE CHARACTERISTICS: Demographic characteristics are presented in Table 1. There were no significant differences in demographic characteristics between the intervention and control groups. In both groups, most participants were males, professionals, and university graduates with no chronic disease. 10.1371/journal.pone.0097167.t001Table 1BODY.BASELINE CHARACTERISTICS OF PARTICIPANTS IN THE INTERVENTION AND CONTROL GROUPS.: Intervention group (N = 381) Control group (N = 381) p * n (%) Average (SD) n (%) Average (SD) Age (years) 38.0(9.2) 37.2(8.8) 0.24 Gender 0.33 Male 325(85.3) 314(82.4) Female 56(14.7) 67(17.6) Marital status 0.47 Never married 161(42.3) 150(39.4) Married 212(55.6) 226(59.3) Divorced or bereaved 8(2.1) 5(1.3) Occupation 0.25 Manager 93(24.4) 79(20.7) Professional 256(67.2) 278(73.0) Clerical 18(4.7) 14(3.7) Production 3(0.8) 3(0.8) Sales 3(0.8) 5(1.3) Others 8(2.1) 2(0.5) Education 0.59 High school 43(11.3) 34(8.9) Some collage 60(15.7) 70(18.4) University 245(64.3) 242(63.5) Graduate school 33(8.7) 35(9.2) Chronic disease 0.56 Yes 38(10.0) 44(11.5) No 343(90.0) 337(88.5) *t-test or χ2 test. BODY.EFFECTS OF THE ICBT PROGRAM ON EACH OUTCOME VARIABLE: Table 2 shows the means and SDs of the outcome variables at baseline, three-month, and six-month follow-ups in the intervention and control groups. Table 3 shows the estimated effects of the iCBT program on the outcome variables based on the mixed-model analyses as well as the effect sizes (Cohen's d). The iCBT program showed a significant effect on BDI-II (t = −1.99, p<0.05). The effect sizes were small. For BDI-II, the effect size was −0.14 (95% CI: −0.30 to 0.02) at the 3-month follow-up and −0.16 (95% CI: −0.32 to 0.00) at the 6-month follow-up. 10.1371/journal.pone.0097167.t002Table 2BODY.MEANS (SDS) OF OUTCOME VARIABLES AT BASELINE, 3- AND 6-MONTH FOLLOW-UP IN THE INTERVENTION AND CONTROL GROUPS FOR THE WHOLE SAMPLE.: Intervention Control Baseline (N = 381) 3 month (N = 270) 6 month (N = 272) Baseline (N = 381) 3 month (N = 336) 6 month (N = 320) mean (SD) mean (SD) mean (SD) mean (SD) mean (SD) mean (SD) BDI-II 11.9 (8.0) 10.7 (8.6) 11.3 (9.6) 11.8 (8.0) 11.7 (8.3) 12.1 (8.7) K6 5.6 (4.6) 5.6 (4.6) 5.7 (4.8) 5.6 (4.7) 5.8 (4.7) 6.4 (5.1) DAS 88.0 (21.5) 87.4 (21.7) 84.9 (23.3) 87.4 (20.9) 88.5 (20.9) 87.0 (22.8) Overtime hours during the past month 32.1 (29.5) 32.4 (28.6) 35.8 (31.1) 29.0 (25.9) 30.2 (21.8) 35.5 (27.0) Scores of knowledge Stress management 1.6 (0.9) 2.1 (0.8) 2.2 (0.9) 1.7 (1.0) 1.8 (0.9) 1.8 (0.9) Cognitive restructuring 1.5* (0.9) 2.0 (0.8) 2.1 (0.9) 1.6 (0.9) 1.7 (0.9) 1.8 (0.9) Assertive communication 1.5 (0.9) 1.9 (0.8) 1.9 (0.8) 1.6 (0.9) 1.7 (0.9) 1.7 (0.9) Problem-solving 1.6 (0.8) 1.9 (0.8) 1.9 (0.8) 1.6 (0.9) 1.7 (0.8) 1.7 (0.9) Relaxation training 1.5 (0.9) 1.9 (0.8) 2.0 (0.8) 1.5 (0.9) 1.6 (0.9) 1.7 (0.9) Scores of self-efficacy Stress management 1.8 (1.0) 2.1 (0.9) 2.0 (1.0) 1.9 (1.0) 1.8 (1.0) 1.9 (1.0) Cognitive restructuring 1.7* (1.0) 1.9 (0.9) 1.9 (1.0) 1.7 (0.9) 1.7 (0.9) 1.7 (1.0) Assertive communication 1.4 (0.9) 1.7 (0.8) 1.6 (0.9) 1.4 (0.9) 1.5 (0.9) 1.6 (0.9) Problem-solving 1.7 (0.8) 1.8 (0.8) 1.8 (0.8) 1.7 (0.9) 1.7 (0.8) 1.7 (0.9) Relaxation training 1.5 (0.9) 1.7 (0.9) 1.8 (0.9) 1.5 (0.9) 1.5 (0.9) 1.6 (1.0) Note: BDI-II =  Beck Depression Inventory II, K6 =  Kessler's psychological distress scale, DAS =  Japanese version of the Dysfunctional Attitude Scale 24. * The number of participants was 380 in intervention group on Knowledge of cognitive restructuring and Efficacy of cognitive restructuring at baseline. 10.1371/journal.pone.0097167.t003Table 3BODY.EFFECT OF THE INTERNET-BASED COMPUTERIZED COGNITIVE BEHAVIORAL THERAPY (ICBT) PROGRAM ON PRIMARY AND SECONDARY OUTCOME VARIABLES FOR THE WHOLE SAMPLE.: Estimates of fixed effects Cohen's d Effect SE df t p 95% CI T2-T1 95% CI T3-T1 95% CI BDI-II −0.51 0.26 621.35 −1.99 <0.05 −1.02 to −0.01 −0.14 −0.30 to 0.02 −0.16 −0.32 to 0.00 K6 −0.29 0.17 646.59 −1.72 0.09 −0.63 to 0.04 −0.01 −0.17 to 0.15 −0.14 −0.30 to 0.02 DAS −1.69 0.69 625.86 −2.43 0.02 −3.05 to −0.32 −0.11 −0.27 to 0.05 −0.20 −0.36 to −0.04 Overtime hours during the past month −1.73 1.21 652.18 −1.43 0.15 −4.10 to 0.64 −0.04 −0.20 to 0.12 −0.13 −0.29 to 0.03 Improvement of knowledge Stress management 0.22 0.04 650.68 5.85 <0.01 0.15 to 0.29 0.44 0.28 to 0.60 0.45 0.29 to 0.61 Cognitive restructuring 0.20 0.04 651.23 5.17 <0.01 0.12 to 0.27 0.45 0.29 to 0.61 0.40 0.24 to 0.56 Assertive communication 0.17 0.03 643.86 4.86 <0.01 0.10 to 0.23 0.43 0.27 to 0.59 0.40 0.24 to 0.56 Problem-solving 0.14 0.03 635.19 4.24 <0.01 0.08 to 0.21 0.55 0.39 to 0.71 0.34 0.17 to 0.50 Relaxation training 0.20 0.04 646.42 5.53 <0.01 0.13 to 0.27 0.46 0.29 to 0.62 0.44 0.28 to 0.60 Improvement of self-efficacy Stress management 0.10 0.04 633.62 2.72 0.01 0.03 to 0.17 0.35 0.19 to 0.51 0.23 0.07 to 0.39 Cognitive restructuring 0.11 0.04 638.42 2.83 <0.01 0.03 to 0.18 0.33 0.17 to 0.49 0.23 0.07 to 0.39 Assertive communication 0.09 0.03 649.87 2.51 0.01 0.02 to 0.15 0.34 0.18 to 0.50 0.21 0.04 to 0.37 Problem-solving 0.06 0.03 646.84 1.82 0.07 0.00 to 0.12 0.29 0.13 to 0.45 0.17 0.00 to 0.33 Relaxation training 0.13 0.04 651.05 3.39 <0.01 0.05 to 0.21 0.32 0.16 to 0.48 0.28 0.11 to 0.44 Note: BDI-II =  Beck Depression Inventory II, K6 =  Kessler's psychological distress scale, DAS =  Japanese version of the Dysfunctional Attitude Scale 24, T1 =  baseline, T2 =  3-month follow-up, T3 =  6-month follow-up. BODY.SECONDARY OUTCOMES: The iCBT program showed a significant effect on DAS (t = -2.43, p = 0.02) and all knowledge and efficacy variables (p<0.05) except for the efficacy of problem solving. The program showed a marginally statistically significant effect on K6 (t = −1.72, p = 0.09) and efficacy of problem-solving (t = 1.82, p = 0.07). Effect sizes for K6, DAS, and improvement of self-efficacy were small, while effect sizes for knowledge variables were medium. BODY.NNT FOR IMPROVING SUBTHRESHOLD DEPRESSION: Among respondents at the 3-month follow-up, 32.1% and 20.2% had a remission from subthreshold depression defined by BDI-II and K6, respectively. At the 6-month follow-up, 30.1% and 19.7% of respondents had a remission from subthreshold depression on BDI-II and K6, respectively. No significant difference was observed in the remission based on BDI-II between the intervention and control groups: relative risk (RR) of having subthreshold depression in the intervention group were 0.79 (p = 0.22) at the 3-month follow-up and 0.77 (p = 0.20) at the 6-month follow-up; the NNTs to achieve one remission from subthreshold depression were 14 at the 3-month follow-up and 13 at the 6-month follow-up. The probability of the remission based on K6 was not significantly different between the groups at the 3-month follow-up (RR = 1.02, p = 0.94) and was marginally significantly higher in the intervention group than in the control group at the 6-month follow-up (RR = 0.63, p = 0.06); the NNTs were -279 at the 3-month follow-up and 12 at the 6-month follow-up. BODY.SUBGROUP ANALYSES: BODY.RESPONDENTS WITH HIGH PSYCHOLOGICAL DISTRESS: The subgroup analysis was conducted for participants who scored 5 or more on K6 at the baseline. Table 4 shows the means and SDs of outcome variables at baseline, 3-month, and 6-month follow-ups for the intervention and control groups who scored 5 or more on the K6 at the baseline (N = 194 for each group). Table 5 shows the estimated effects of the iCBT program on outcomes variables as well as the effect sizes (Cohen's d). The iCBT program showed a significant effect on BDI-II (t = −2.12, p = 0.04). The effect size for BDI-II was small, −0.16 at the 3-month follow-up and −0.25 at the 6-month follow-up. 10.1371/journal.pone.0097167.t004Table 4BODY.MEANS (SDS) OF OUTCOME VARIABLES AT BASELINE, 3- AND 6-MONTH FOLLOW-UP IN THE INTERVENTION AND CONTROL GROUPS: ANALYSIS OF PARTICIPANTS WITH HIGH PSYCHOLOGICAL DISTRESS AT BASELINE (K6≧5).: Intervention Control Baseline (N = 194) 3 month (N = 125) 6 month (N = 130) Baseline (N = 194) 3 month (N = 167) 6 month (N = 160) mean (SD) mean (SD) mean (SD) mean (SD) mean (SD) mean (SD) BDI-II 16.9 (7.3) 15.4 (8.9) 15.7 (10.2) 16.3 (8.0) 15.8 (8.2) 16.2 (8.3) K6 9.2 (3.6) 8.3 (4.6) 8.3 (5.0) 9.2 (4.7) 8.2 (4.6) 9.0 (4.7) DAS 95.5 (19.8) 95.3 (19.8) 92.7 (22.4) 92.9 (20.9) 94.2 (19.9) 94.0 (21.1) Overtime hours during the past month 33.9 (31.3) 33.9 (29.0) 40.8 (33.9) 28.5 (25.9) 30.3 (21.1) 35.5 (25.0) Scores of knowledge Stress management 1.4 (0.8) 1.9 (0.7) 2.0 (0.8) 1.5 (0.9) 1.6 (0.8) 1.7 (0.8) Cognitive restructuring 1.3* (0.8) 1.8 (0.7) 1.8 (0.8) 1.4 (0.9) 1.5 (0.7) 1.6 (0.9) Assertive communication 1.3 (0.8) 1.9 (0.8) 1.7 (0.8) 1.4 (0.8) 1.5 (0.8) 1.5 (0.8) Problem-solving 1.4 (0.8) 1.9 (0.7) 1.7 (0.8) 1.4 (0.8) 1.5 (0.7) 1.6 (0.8) Relaxation training 1.2 (0.8) 1.8 (0.8) 1.8 (0.8) 1.3 (0.8) 1.3 (0.7) 1.5 (0.8) Scores of self-efficacy Stress management 1.5 (0.9) 1.8 (0.8) 1.7 (0.9) 1.5 (1.0) 1.5 (0.8) 1.5 (0.9) Cognitive restructuring 1.3* (0.9) 1.7 (0.8) 1.5 (0.9) 1.3 (0.8) 1.4 (0.8) 1.4 (0.9) Assertive communication 1.1 (0.8) 1.5 (0.8) 1.3 (0.8) 1.3 (0.8) 1.2 (0.8) 1.3 (0.8) Problem-solving 1.4 (0.7) 1.6 (0.7) 1.6 (0.8) 1.4 (0.8) 1.4 (0.8) 1.5 (0.8) Relaxation training 1.1 (0.8) 1.5 (0.8) 1.5 (0.9) 1.2 (0.8) 1.3 (0.7) 1.3 (0.8) Note: BDI-II =  Beck Depression Inventory II, K6 =  Kessler's psychological distress scale, DAS =  Japanese version of the Dysfunctional Attitude Scale 24. * The number of participants was 193 in intervention group on Knowledge of cognitive restructuring and Efficacy of cognitive restructuring at baseline. 10.1371/journal.pone.0097167.t005Table 5BODY.EFFECT OF THE ICBT PROGRAM ON PRIMARY AND SECONDARY OUTCOME VARIABLES: ANALYSIS OF PARTICIPANTS WITH HIGH PSYCHOLOGICAL DISTRESS AT BASELINE (K6≧5).: Estimates of fixed effects Cohen's d Effect SE df t p 95% CI T2–T1 95% CI T3–T1 95% CI BDI-II −0.88 0.42 308.23 −2.12 0.04 −1.70 to −0.06 −0.16 −0.40 to 0.07 −0.25 −0.48 to −0.02 K6 −0.39 0.27 542.61 −1.45 0.15 −0.92 to 0.14 −0.03 −0.26 to 0.20 −0.19 −0.42 to 0.05 DAS −2.33 0.99 295.92 −2.36 0.02 −4.28 to −0.38 −0.13 −0.36 to 0.11 −0.27 −0.51 to −0.04 Overtime hours during the past month −1.19 1.61 345.91 −0.74 0.46 −4.35 to 1.98 −0.08 −0.32 to 0.15 −0.11 −0.34 to 0.12 Improvement of knowledge Stress management 0.18 0.05 323.55 3.66 <0.01 0.08 to 0.28 0.43 0.20 to 0.67 0.39 0.15 to 0.62 Cognitive restructuring 0.20 0.05 320.72 3.84 <0.01 0.10 to 0.30 0.63 0.39 to 0.87 0.44 0.20 to 0.67 Assertive communication 0.12 0.04 570.27 3.14 <0.01 0.05 to 0.20 0.61 0.37 to 0.84 0.29 0.05 to 0.52 Problem-solving 0.16 0.05 309.67 3.42 <0.01 0.07 to 0.25 0.71 0.47 to 0.95 0.38 0.15 to 0.62 Relaxation training 0.24 0.05 319.64 4.66 <0.01 0.14 to 0.33 0.66 0.42 to 0.89 0.53 0.29 to 0.76 Improvement of self-efficacy Stress management 0.10 0.05 310.38 2.09 0.04 0.01 to 0.20 0.39 0.16 to 0.63 0.24 0.01 to 0.48 Cognitive restructuring 0.09 0.05 312.70 1.66 0.10 −0.02 to 0.19 0.41 0.18 to 0.65 0.21 −0.03 to 0.44 Assertive communication 0.14 0.05 320.71 2.96 <0.01 0.05 to 0.23 0.57 0.33 to 0.81 0.34 0.10 to 0.57 Problem-solving 0.07 0.05 315.91 1.60 0.11 −0.02 to 0.16 0.38 0.15 to 0.62 0.20 −0.03 to 0.43 Relaxation training 0.16 0.04 622.71 3.71 <0.01 0.07 to 0.24 0.40 0.17 to 0.64 0.34 0.10 to 0.57 Note: BDI-II =  Beck Depression Inventory II, K6 =  Kessler's psychological distress scale, DAS =  Japanese version of the Dysfunctional Attitude Scale 24, T1 =  baseline, T2 =  3-month follow-up, T3 =  6-month follow-up. The iCBT program showed a significant effect on DAS (t = −2.36, p = 0.02), all knowledge variables, efficacy of stress management, assertive communication, and relaxation training (p<0.05). While the effect sizes of DAS and self-efficacy were small, those for variables on knowledge were medium. BODY.PROCESS EVALUATION: Table 6 shows the process evaluation indicators of the iCBT programs only for the intervention group. Most (89%) participants in the intervention group completed Lesson 1, and 65% submitted their homework after completing this lesson. The proportion of those who completed lessons and submitted homework gradually decreased during the later lessons. About two thirds of the intervention group completed all six lessons, while only a quarter of them submitted all six homework assignments. The average number of lessons that the respondents received was 4.5. The average number of homework assignments submitted was 2.7. Three quarters of the intervention group completed at least three lessons, and about half (45%) submitted at least three homework assignments. 10.1371/journal.pone.0097167.t006Table 6BODY.PROGRESS OF LEARNING IN THE ICBT PROGRAM AMONG THE INTERVENTION GROUP PARTICIPANTS: THE WHOLE SAMPLE.: Contents Completers of lessons Submitters of homework Lesson 1 Learning about stress 340(89.2%) 248(65.1%) Lesson 2 Knack for self case formulation based on CB model 317(83.2%) 193(50.7%) Lesson 3 Try cognitive restructuring part 1 291(76.4%) 176(46.2%) Lesson 4 Try cognitive restructuring part 2 272(71.4%) 149(39.1%) Lesson 5 Knack for communication 258(67.7%) 127(33.3%) Lesson 6 How to solve your problem effectively 247(64.8%) 117(30.7%) All six lessons 247(64.8%) 93(24.4%) Average number of respondents who completed per lesson (proportion in to the total sample, %). 287.5(75.5%) Average number of respondents who submitted homework per lesson (proportion to the total sample, %). 168.3(44.2%) Average number of sessions completed per respondent. 4.53 Average number of homework submitted per respondent. 2.65 The number of respondents who completed more than 3 lessons (proportion to the total sample). 291(76.4%) The number of respondents who submitted homework more than 3 times (proportion to the total sample). 173(45.4%) BODY.DISCUSSION: The present RCT examined the effects of a newly developed iCBT program for improving the symptoms of depression and other outcomes at three- and six-month follow-ups among workers employed in private companies in Japan. In the total sample, the iCBT program showed a significant intervention effect on depression, as measured by BDI-II, with the effect sizes of −0.16 at the six-month follow-up. The iCBT program also showed a marginally statistically significant intervention effect on psychological distress measured by K6 and significant intervention effects on improving dysfunctional attitude, knowledge, and self-efficacy of most components of CBT. This iCBT program may effectively improve depressive symptoms among workers with the universal approach (i.e., targeting the whole working population), even though the effect size may be modest. To our knowledge, the present study first demonstrated that an Internet-based computerized CBT was effective in improving depression in a non-clinical working population. In the intervention group, BDI-II scores improved at the three-month follow-up and returned to the baseline level at the six-month follow-up. In the control group, BDI-II scores stayed at almost the same level at the three-month follow-up and increased slightly by the six-month follow-up. This difference yielded effect sizes of −0.14 and −0.16 on BDI-II scores at the three- and six-month follow-ups, respectively. After the baseline survey, one of the two companies that participated in the study merged with another company. The observed increase in depression in the control group was partly attributable to the increased workload associated with the organizational change. The current iCBT program seems to improve depression to some extent under severe working conditions. With consideration for the fact that an iCBT can be delivered to many workers at a low cost, this iCBT may be a promisingly cost-effective approach for improving depression among workers. The effect size of the current iCBT program for improving depression was small (around 0.15). Grime (2004) reported that the effect sizes (Cohen's d) of CCBT on depression among workers who recently had stress-related absenteeism were −0.25 (p = 0.04) at one month after the end of intervention and −0.1 (p = 0.56) at the three-month follow-up [18]. The current iCBT program might be effective in improving depression among workers to a similar or greater extent compared to the ordinary CCBT program. The small effect sizes in the present study could be attributable to the non-clinical sample and the lower degree of therapist involvement in this iCBT program [16], [17]. In fact, the iCBT program showed a better intervention effect on BDI-II, when the analysis was limited to those who have high depression score at baseline (−0.16 and −0.25 at the three- and six-month follow-ups, respectively), which is concordant with a previous meta-analysis that the effect of CBT programs in preventing major depressive disorder was greater for selective and indicated interventions than the universal one [33]. In the present study, the NNTs to achieve one remission from subthreshold depression were 14 at the 3-month follow-up and 13 at the 6-month follow-up for BDI-II. These were −279 at the 3-month follow-up and 12 at the 6-month follow-up for K6. These figures were larger than those of the previously reported NNTs of iCBT for improving depression among patients with major depressive disorder (2 to 4), when compared with TAU or waiting list [32]. However, these were lower (thus better) than a NNT that reported a RCT using the automated e-mails containing general information and advice about self-help strategies for depression to achieve one remission from subthreshold depression (26) [38]. The NNTs of the present iCBT program fall between these studies of clinical and non-clinical populations. The iCBT program showed a significant intervention effect on dysfunctional attitude measured by the DAS in terms of knowledge and efficacy of most CBT components except for self-efficacy of problem solving. Dysfunctional attitude is one of major targets of cognitive restructuring, a component of the current program. The present findings are consistent with previous ones that a CCBT program was effective in improving dysfunctional attitudes among medication-free participants with major depressive disorder [39], while the present study observed a larger effect size than that of the previous study (0.5). The effect sizes for knowledge were better than the one reported by a previous worksite-based study [40]. In addition, improved levels of knowledge and self-efficacy were maintained at the six-month follow-up. Downloadable PDF summaries of sessions of the iCBT program may have helped participants maintain their knowledge and self-efficacy. This may also be a benefit of using Manga story as a part of the iCBT program, while we did not systematically assess it. The process evaluation of the iCBT program indicated that proportions of those who completed lessons and submitted homework decreased for the later lessons. About two-thirds of the intervention group completed all six lessons, while only a quarter of them submitted all six homework assignments. The completion rates of self-help programs without clinician contact were generally low [13]. The average number of completed lessons per respondent (4.5) in this six-lesson iCBT program was very close to that of a previous seven-chapter self-help iCBT program [41], [42]. The low rates of completing the lessons and submitting homework may have resulted in a smaller effect size in this study. It would be critical to increase completion rates to improve the effect of an iCBT program. BODY.LIMITATIONS: This study has several limitations. First, participants were recruited from two IT companies in Japan. Most participants were males, working as professionals, and university graduates. They had their own PCs in their offices or homes. The participants were also supposed to have experience using a PC and studying through online programs. Higher education level may also help participants learn from the iCBT program. The generalization of the present findings to the general working population is limited. Second, the rate of completing lessons and homework was low. Only 93 participants submitted all homework assignments, and the average number of homework assignments submitted was 2.7 per respondent. This may weaken the findings. Third, the dropout rates in the present study were 29.1% and 28.6% at the three- and six-month follow-ups, respectively. In their systematic review, Kaltenthaler et al. (2008) reported that average CCBT dropout rates were 18–35% [43]. Wantland et al. (2004) reported that an average dropout rate was 21% [44]. The dropout rates in the present study were within the range. However, the dropouts may cause a selection bias, particularly if the intervention group participants with higher levels of depression were more likely to quit the program. Fourth, the present study provided the participants in the control group with e-mails providing stress management tips. This may weaken the intervention effect. Fifth, there was the possibility that participants in the control group could have information about the iCBT program from participants in the intervention group at same workplace. This contamination may weaken the intervention effect. Sixth, all outcomes in the present study were measured by self-report, which may be affected by the perception of participants or situational factors at work. A further RCT should be conducted to examine whether the iCBT program is effective in a larger sample of workers with diverse characteristics, particularly in terms of occupation and education. BODY.SUPPORTING INFORMATION: Checklist S1CONSORT 2010 checklist of information to include when reporting a randomized trial. (DOCX) Click here for additional data file. Protocol S1The original protocol in Japanese. (DOCX) Click here for additional data file. Protocol S2English translation of the original Japanese protocol. (DOCX) Click here for additional data file.

TITLE: A cluster randomized controlled trial of lay health worker support for prevention of mother to child transmission of HIV (PMTCT) in South Africa ABSTRACT.BACKGROUND: We evaluate the impact of clinic-based PMTCT community support by trained lay health workers in addition to standard clinical care on PMTCT infant outcomes. ABSTRACT.METHODS: In a cluster randomized controlled trial, twelve community health centers (CHCs) in Mpumalanga Province, South Africa, were randomized to have pregnant women living with HIV receive either: a standard care (SC) condition plus time-equivalent attention-control on disease prevention (SC; 6 CHCs; n  = 357), or an enhanced intervention (EI) condition of SC PMTCT plus the “Protect Your Family” intervention (EI; 6 CHCs; n  = 342). HIV-infected pregnant women in the SC attended four antenatal and two postnatal video sessions and those in the EI, four antenatal and two postnatal PMTCT plus “Protect Your Family” sessions led by trained lay health workers. Maternal PMTCT and HIV knowledge were assessed. Infant HIV status at 6 weeks postnatal was drawn from clinic PCR records; at 12 months, HIV status was assessed by study administered DNA PCR. Maternal adherence was assessed by dried blood spot at 32 weeks, and infant adherence was assessed by maternal report at 6 weeks. The impact of the EI was ascertained on primary outcomes (infant HIV status at 6 weeks and 12 months and ART adherence for mothers and infants), and secondary outcomes (HIV and PMTCT knowledge and HIV transmission related behaviours). A series of logistic regression and latent growth curve models were developed to test the impact of the intervention on study outcomes. ABSTRACT.RESULTS: In all, 699 women living with HIV were recruited during pregnancy (8–24 weeks), and assessments were completed at baseline, at 32 weeks pregnant (61.7%), and at 6 weeks (47.6%), 6 months (50.6%) and 12 months (59.5%) postnatally. Infants were tested for HIV at 6 weeks and 12 months, 73.5% living infants were tested at 6 weeks and 56.7% at 12 months. There were no significant differences between SC and EI on infant HIV status at 6 weeks and at 12 months, and no differences in maternal adherence at 32 weeks, reported infant adherence at 6 weeks, or PMTCT and HIV knowledge by study condition over time. ABSTRACT.CONCLUSION: The enhanced intervention administered by trained lay health workers did not have any salutary impact on HIV infant status, ART adherence, HIV and PMTCT knowledge. Trial registration clinicaltrials.gov: number NCT02085356 BODY.BACKGROUND: Of the 2.1 million children suffering from HIV globally, most live in Sub-Saharan Africa and have contracted the virus from their HIV-infected mothers [1]. Mother-to-child transmission (MTCT) is the direct transmission of HIV to infants by HIV-infected mothers during or after the gestation period, childbirth, or breastfeeding [2]. The prevalence of MTCT of HIV in South Africa (SA) by 4–8 weeks postnatal in 2012/13 was 2.6%, with Mpumalanga Province having an MTCT rate of 1.5% [3], and within the same cohort, cumulative MTCT was at 3 months (2.7%), 6 months (3.5%), 9 months (3.7%), 12 months (3.9%) and 18 months (4.3%) [4]. In the latest National Antenatal Sentinel HIV Prevalence Survey (2013) among women 15–49 years the national HIV prevalence was 29.7%, and in Mpumalanga province 37.5% [5]. These high rates of HIV in rural areas often been attributed to supply and staff shortages, as well as to limited access to care [6]. For the prevention of mother-to-child-transmission (PMTCT) of HIV, the World Health Organization (WHO) proposes that all pregnant women, regardless of CD4 count, be given ART for life (Option B+ ART policy), a policy that was adopted by South Africa in 2015. For mothers who breastfeed, daily prophylaxis must be administered to the child for the first 6 weeks of life [7]. To test for HIV infant status, a DNA polymerase chain reaction (PCR) is used to measure levels of viral DNA in the infant’s blood. Virologic PCRs for high-risk infants must be done at birth, 14–21 days postnatal, 1–2 months postnatal, 4–6 months postnatal [8], and 9 months postnatal if infants exhibit symptoms later on [9]. Risk factors associated with MTCT include low HIV knowledge, non-adherence to ART, lower education, HIV stigma, psychological repercussions of being diagnosed with HIV [10], lack of male partner support, and low medication infant dosing [10, 11]. Non-adherence to ART is suspected to occur at an alarming rate during breast-feeding [12, 13], as MTCT rates increase from 4.7% at 6 weeks postnatal to 8.9% at the cessation of breastfeeding [12]. Without any intervention, MTCT is estimated to be between 14 and 45% [14], but with the use of ARTs, caesarean section births, and avoidance of breastfeeding, can be lowered to less than 5% [15, 16]. Previous successful interventions for PMTCT in SA have utilized mother-to-mother peer mentoring and cognitive behavioral interventions (CBI) to increase follow-up appointments, HIV knowledge, and reductions in psychological distress [17]. Other studies have found that improving access to care by increasing the availability of prenatal HIV counselors is an effective PMTCT intervention, and that failure to obtain postnatal care could be improved by increasing hospital stays post-delivery, as well as by follow-up visits by community health workers [18]. An overall understanding of PMTCT by both mothers and the community appears necessary for interventions to be effective [19]. To fully benefit from the PMTCT protocol, HIV-infected pregnant women should be retained in 90% of the steps of the PMTCT cascade, including initiation of maternal antiretroviral (ARV) drugs or therapy, initiation of infant ARV, and infant HIV testing [20, 21]. However, considerable challenges exist to attaining these cascade goals [22]. A recent review of interventions to improve PMTCT utilization [23] indicated that mobile phone-based interventions were associated with increased uptake of early infant diagnosis of HIV at around 6 weeks postpartum and male partner involvement in PMTCT was associated with reductions in infant HIV infection, while studies grounded in psychological interventions failed to increase ARV/ART uptake among HIV-infected pregnant and/or breastfeeding women and to enhance infant HIV testing. Despite the availability of an effective PMTCT treatment protocol, guidelines designed for PMTCT, and successful reductions in transmission, uptake of all elements of PMTCT in rural South Africa remains suboptimal [24]. The ‘Protect Your Family’ project is an ongoing clinical trial designed to enhance uptake of PMTCT elements pre- and postpartum, with the goal of further reducing vertical transmission rates in rural South Africa. Due to the higher rates of MTCT and reduced resources, the primary objective of the current study was to test whether this behavioural intervention delivered by lay health workers in rural South Africa could increase maternal HIV/PMTCT knowledge and reduce HIV transmission to infants. BODY.METHODS: BODY.STUDY DESIGN: This study was a clinic-randomized controlled trial using a 1  ×  2  ×  5 comparison: women ×  condition (experimental or control)  ×  time (assessments given at baseline, 32 weeks pregnant, and 6 weeks, 6 months and 12 months postpartum). In addition to assessments, participants attended three group and one individual counselling intervention (or time-equivalent control) sessions prior to birth, and two individual counselling sessions postpartum [25]. BODY.PRINCIPLES FOR RECRUITMENT: BODY.PUBLIC COMMUNITY HEALTH CENTERS (CHCS): All CHCs from the Gert Sibande and Nkangala Districts in the Mpumalanga province were reviewed in consultation with the Provincial Department of Health. Eligible CHCs clinics met South African criteria for PMTCT sites, including on-site daily HIV counselling and testing (HCT), ART distribution and CD4 T cell testing, ante and postnatal counseling on infant feeding, infant HIV testing, two or more trained PMTCT staff and two counselors and a support group for HIV-infected mothers and pregnant women. Twelve CHCs were randomly assigned as intervention sites or standard of care sites, stratified by antenatal care clinic case load in the upper 50th percentile of MTCT rates at the onset of the study (> 13%). BODY.PREGNANT WOMEN LIVING WITH HIV: Eligible women were HIV-infected pregnant women having a primary male partner; women were between 8 and 24 weeks pregnant, the typical time of entry into antenatal care, and aged 18 years or older. For the purposes of the study, primary male partners were defined as husband, current baby’s father, current male sexual partner or trusted male friend actively involved in the mother’s life. Eligible women agreeing to participate were enrolled following provision of informed consent; male partners were not enrolled. Those identified as actively psychotic (auditory or visual hallucinations) or intoxicated (under the influence of alcohol of illegal drugs) were not eligible and were referred for treatment. There were no exclusions based on literacy as all assessments were administered using an audio computer assisted self-interview (ACASI) system. BODY.RANDOMIZATION: The twelve CHCs were matched in a 1:1 ratio according to patient census, average ANC volume, and MTCT rates; one clinic in each pair was randomly assigned to the experimental or control condition using a computer program written by the data manager. The matched clinics were then assigned to the opposite condition. The randomization process was carried out by four people. The first conducted the computer-generated randomization assignments stratified by clinic size, i.e., selected a seed for the random number generator, ran the program, and completed the table of condition assignments. The second implemented the assignments, providing a table of all clinic site assignments to study personnel. The third activated each intervention site individually, and the fourth activated each control site individually. BODY.BLINDING: This was a double blind study. Following randomization, clinic sites were activated individually, and training for clinic study staff was conducted by condition. Study staff conducting the assessments and conditions were blind to their clinic randomization status, and both clinic staff and participants were blinded to condition. Data analysis to evaluate study outcomes were blinded to clinic condition. Only the Human Sciences Research Council (HSRC) project study staff activating and overseeing the sites and the intervention and control condition trainers for study staff were aware of site assignment. BODY.INTERVENTIONS: BODY.INTERVENTION CONDITION: IE participants receive the PMTCT standard of care (according to the Option B treatment protocol, ARVs will continue to be provided to the mother after the cessation of breastfeeding only if the mothers’ health requires it) plus three prenatal weekly 2-h group sessions (between five and seven participants) followed by one individual counselling session and 2 monthly individual counselling sessions (one prenatal, two postpartum) led by study-trained clinic staff. The ‘Protect Your Family’ intervention is a manualized, closed, structured behavioral risk-reduction program. The intervention targeted prevention of vertical transmission, adherence to PMTCT and medication use, HIV testing of family members, prevention of HIV transmission and stigma, serostatus disclosure, partner communication, intimate partner violence (IPV) reduction, safe infant feeding, safer conception, family planning and dual method sexual barrier use. Intervention elements have been previously described [25]. BODY.CONTROL CONDITION: Standard care condition participants received the PMTCT standard of care plus a time-equivalent, group-administered video presentation on childhood disease prevention (e.g., measles, diarrhoea management, dysentery and dehydration, and immunizations and vaccinations) in three group sessions, followed by one individual and two couple or individual women’s sessions on disease prevention. BODY.TRAINING OF STUDY STAFF MEMBERS AND INTERVENTION QUALITY ASSURANCE: Study staff at all CHC sites underwent a 3-day formal training on the study protocol, informed consent, protection of human subjects, recruitment, assessment and use of ACASI technology, with an in-depth review of the meaning of each item in the assessment instruments presented by ACASI, and presented by the UM and HSRC investigators. Following the training, all staff received ongoing biweekly supervision by HRSC investigators at their CHC sites on the study protocol for data collection. Enhanced intervention condition staff attended a 5-day training course that included an intensive review of the ‘Protect Your Family’ intervention manual, the PMTCT protocol and use of cognitive behavioral (CB) intervention strategies in the intervention, as well as how to manage sensitive issues (e.g., serostatus disclosure, IPV, gender dynamics, sexual risk reduction and safer conception practices). EI condition staff also received ongoing guided training and practice on the intervention under the supervision of the intervention coordinator, who acted as leader and then co-leader of the intervention at each EI CHC site for the first two cohorts, and annual training over 3 years. Thus, each IE clinic staff member conducted two sequences of group sessions and individual counselling sessions under the supervision of the HSRC coordinator. Intervention fidelity was continually assessed using audio recordings of intervention sessions and interventionist checklists that were reviewed by the intervention coordinator monthly; a randomly selected sample of 10% of the total number of sessions was transcribed by study staff using headphones in private rooms at the HSRC offices and reviewed by the senior HSRC staff trainer for fidelity. Intervention session checklists were reviewed by UM study staff in collaboration with HSRC staff. Standard care condition staff received an identical 1-day training session on the use of ACASI technology and a 4-h orientation to the protocol to enable them to conduct time-equivalent group sessions comprised of childhood disease prevention and adult health hazard videotapes (e.g., measles, diarrhoea management, dysentery and dehydration, and immunizations and vaccinations). Fidelity information was not collected for the control arm, as the intervention consisted of the presentation of a video recording. Control condition providers were interviewed during regularly occurring visits and annual training to confirm the ongoing provision of video recordings. BODY.OUTCOME EVALUATION: Primary outcomes included infant HIV status and ART adherence for mothers and infants. Infant HIV status was assessed via PCR at 6 weeks postnatal as part of the South African PMTCT standard of care, and the results were collected from the Road to Health booklet (a patient-held record of the child’s well-being) or clinic records by an external research assistant. A second HIV test was administered at 12 months as part of study participation, and assessed via DNA PCR. Maternal adherence to antiretrovirals was assessed by dried blood spot using procedures and strategies previously described [26]. Maternally reported infant adherence to nevirapine was assessed using a Visual Analog Scale, in which participants rated the level of adherence to infant medication on a scale of 0 (took none of the medication) to 1 (took half of the medication) to 2 (took all of the medication) for each day in the past week (3). Participant responses consistent with having missed a dose in the past 7 days were considered nonadherent, whereas participants who reported having missed zero doses were considered adherent. Secondary outcomes included HIV and PMTCT knowledge, and was assessed using an adaptation of the AIDS-Related Knowledge Test; items reflect information about HIV transmission, reinfection with resistant virus, condom use and PMTCT-specific knowledge [27, 28]. All measures were provided in local languages and had been adapted to the local setting as appropriate. Participants completed study measures in their preferred language (English, Zulu, Sotho) using ACASI to enhance disclosure and reduce bias. BODY.SAMPLE SIZE DETERMINATION: The sample size was determined by a power analysis for the primary outcome of infant HIV status at 6 weeks. Averaged clinic data collected during and following a pilot study in 2012 indicated that approximately 13% of infants would be HIV positive at 6 weeks of age. Using an HIV PCR rate of 13% in the control arm, a power analysis indicated that six sites per condition (six experimental, six control) with an unadjusted sample size of 564 infants would provide 80% power to detect a significant difference between conditions assuming a reduction to 4% in the intervention condition and intracluster correlation coefficients of up to .02 (depending on the two rates) with a two-tailed test at the .05 level [25]. The sample size of 720 pregnant women was based on our experience in the pilot study, from which we anticipate a 16% miscarriage and infant death rate and a 5% attrition rate over 12 months (n = 156 lost; n = 564 retained). BODY.DATA MANAGEMENT AND ANALYSES: Data quality assurance procedures, including reviewing for errors and consistency checks, were completed monthly by the data manager. The quality of biological data was monitored by the site laboratory under the accreditation standards of the South African National Accreditation Systems. The analytic plan consisted of several steps. First, Chi square tests (for categorical variables) and independent t tests (continuous and normally distributed variables) were conducted to examine whether baseline differences on demographic characteristics and outcome variables existed by intervention condition. Second, attrition analysis using binary logistic regression was conducted to compare participants who dropped out after baseline to those included by demographic characteristics [i.e., participants’ age, education level, numbers of children, income, diagnosis with HIV during the pregnancy, HIV positive children, HIV positive partner, disclosure to partner, and marital status (Reference: currently married, living together)]. Third, given the nature of binary primary outcomes (i.e., infant HIV status at 6 weeks and at 12 months, maternal adherence at 32 weeks, and mother-reported infant adherence at 6 weeks), a series of logistic regression models was used to estimate the intervention effects on primary outcomes. To adjust for potential covariate effects, the same baseline covariates that were used for attrition analyses were considered when estimating logistic coefficients. In order to take into account site variations across 12 sites on the associations between intervention effects and primary outcomes, the intervention effects on primary outcomes were adjusted by treating site variation on intervention effects as random variables (i.e., random intercept and random slope model). For model identification, the covariance between random intercept and random slope were fixed to 0. Effect sizes for intervention effects are shown using odds-ratios [29]. Fourth, a latent growth curve model (LGCM) was used to estimate the longitudinal change of secondary outcomes (i.e., HIV knowledge and PMTCT knowledge) over time [30]. The LGCM estimates growth parameters, such as the baseline intercept and longitudinal changes (i.e., trajectories; from baseline to 24 months post baseline period), from data obtained at several measurement points. The method also estimates the mean and the variances of the growth parameters. Consequently, the modeling provided the information to determine how secondary outcomes change during the post baseline period (i.e., trajectories) and if there was individual variation in secondary outcomes trajectories. To adjust for the potential covariate effects on secondary outcome trajectories, the same baseline covariates were included for LGCM that were used to estimate primary outcome models. In this study, time was centered at baseline and evaluated linear growth model across time. In order to take into account the variation of the intervention effect on target trajectories at between-site levels, a multilevel model approach (MLM) was used in the latent growth curve model [31]. For the model identification, the variance of a linear random slope growth factor was fixed to 0 at site levels. The effect sizes of the intervention on growth parameters in HIV knowledge were calculated as the ratio of the difference in the slope means divided by standard deviation of the slope growth factor [32]. 33]. Imputation is a better tool compared to maximum likelihood estimation which has been widely used in the model estimation of clinical research when the dataset contains both categorical and continuous variables [34]. Acock [35] suggested that the common mechanism for missingness may be accounted for by demographic information (e.g., education, race, age, and gender etc.). Therefore, we used baseline demographic variables as covariates when conducting imputation technique (see Table 1). Results were averaged across ten imputed data sets [33, 36].Table 1Baseline demographic and psychosocial characteristics of women (N = 699) Total (N = 699)Control (n = 357)Intervention (N = 342)t, X2, p Sociodemographics  Median age (inter quartile range) 28.0 (8) 28.0 (9) 28.0 (8) .615, .539  Education   0–Grade 9 149 (21.5) 74 (20.8) 75 (22.2)   Grade 10–11 344 (49.6) 171 (48.2) 173 (51.2)   Grade 12 or more 200 (28.6) 110 (31.0) 90 (26.6) 1.60, .449  Relationship status   Married or cohabiting 285 (41.1) 154 (43.4) 131 (38.8)   Having partner, not cohabiting 408 (58.9) 201 (56.6) 207 (61.2) 2.45, .294  Monthly income 1113.3 (2722.0) 1010.0 (1586.3) 1216.5 (3542.7) .97, .330  Number of children   None 140 (20.2) 85 (23.9) 55 (16.3)   One or more 553 (79.8) 270 (76.1) 283 (83.7) 3.62, .143 Health variables  Diagnosed with HIV during this pregnancy 375 (54.1) 195 (54.9) 180 (53.3) .20, .658  HIV status disclosure to partner 402 (59.0) 193 (55.9) 209 (62.2) .22, .642  Partner HIV positive 171 (25.1) 98 (28.4) 73 (21.7) 4.04, .052  Has HIV positive child 29 (5.2) 15 (5.5) 14 (4.9) .10, .756  HIV knowledge, M (SD) 13.8 (3.2) 14.3 (2.9) 13.3 (3.4) 4.01, < .001  PMTCT knowledge, M (SD) 4.6 (1.6) 4.9 (1.5) 4.4 (1.7) 4.12, < .001  Maternal ART adherence (100%) 470 (69.0) 268 (77.7) 202 (60.1) .44, .506 We used a Monte-Carlo simulation in Mplus to conduct a power analysis based on data obtained, acconting for our rates of attrition (Muthén & Muthén, Los Angeles, CA). All conditions were same as previously described, with the exception of specifying 50% attrition rates at 6 weeks for the primary outcome that was used to calculate the sample size a priori: infant HIV status. The results showed that with a sample size of 699 at baseline with attrition rates of 50% at 6 weeks, there is 82% power to detect a significant difference between conditions assuming a reduction to 4% in the intervention condition, which represents a small effect [37]. Based on our results, we believe that total sample size is enough to detect the small intervention effect. Lastly, investigate the intervention effect on both primary and secondary outcomes, we calculated all available effect sizes (i.e., odds-ratios and cohen’s d). Because effect sizes are independent of sample size [37], we believed that the small and non-significant intervention effects were not due to the missing cases. All data analyses were conducted using Mplus (version 7.4) [36]. The principle of the statistical analysis was intention to treat. BODY.RESULTS: BODY.RECRUITMENT AND RANDOMIZATION: Figure 1 summarizes clinic and patient identification, recruitment, randomization, and follow-up numbers. The trial began recruitment from April 2014 to April 2015 and the trial ended in March 2017. As illustrated, 709 eligible pregnant women were identified, 8 declined to participate, and 2 had incomplete data, resulting in 699 patients across 12 community health centers. Participants in community health centers were randomized into six enhanced intervention (EI) community health centers, and six standard care (SC) CHCs, using the clinic as a unit of randomization for 342 participants in the EI condition, and 357 participants in the SC condition (Fig. 1).Fig. 1Flow-chart of clinics and participants in the trial BODY.BASELINE DEMOGRAPHIC AND PSYCHOSOCIAL CHARACTERISTICS OF WOMEN: Women were a median age of 28.0 years (IQR = 8, range 18–46) at baseline, and all women were either married or cohabiting or having a partner and not cohabiting. The majority (79.8%) of the women had one or more children, some (5.2%) had a child living with HIV. Almost half of the women (45.9%) had been diagnosed with HIV prior to the current pregnancy. More than half (59.0%) had disclosed their HIV status to their partner, and 25.1% of their partners were known to be HIV positive. Further description and comparisons by condition are presented in Table 1. The correlations among all study covariates ranged from .008 (between education levels and partner HIV positive) to .446 (between participant’s age and the numbers of children), which were modest. Therefore, collinearity among covariates was not an issue. BODY.ATTRITION ANALYSES: Out of N = 699 women, n = 196 (28.0%) completed all study visits, n = 140 (20.0%) completed four, n = 104 (14.9%) completed three, n = 104 (14.9%) completed two, and n = 155 (22.2%) women completed only the baseline visit. To predict dropout participants after baseline (n = 155, 22.2%), multivariate logistic regression analyses were conducted to compare the key characteristics of the participants in the currents study with participants that dropped out after baseline. Results indicate that participants’ education levels and diagnosis with HIV during the current pregnancy predicted drop out after baseline. That is, participants with higher education levels were less likely to drop out (odds ratio [OR] = .82, p < .01), those having more children were less likely to drop out (OR .81, p < .10), and those having an HIV-infected infant were less likely to drop out (odds ratio [OR] .64, p < .10). No differences were detected by participants’ age, income, partner HIV positive, disclosure to partner, or relationship status. BODY.IMPLEMENTATION INTERVENTION FIDELITY ANALYSIS: BODY.ENHANCED INTERVENTION (EI): Based on a review of audio recordings of 10% of the total number of intervention sessions and interventionist checklists, fidelity analysis of group sessions indicated that clinics provided 58–88% of the intervention elements. Following additional training and supervision, 75–96% of session content was delivered. However, in subsequent individual sessions, fidelity ranged from 47 to 100% of session content was delivered. Ongoing support and training was provided to low fidelity site staff by study trainers and site staff identified as having a high degree of fidelity. Site staff failing to improve over a 12-month period were replaced with new staff who received training and supervision. BODY.PRIMARY OUTCOMES: Results for primary outcomes are presented in Table 2. No primary outcomes were predicted by intervention condition. After controlling for the noted covariates, intervention condition did not predict infant HIV status at 6 weeks (OR 1.24, 95% CI .26, 5.86) or at 12 months (OR .32, 95% CI .00, 122.58). Intervention condition also did not predict maternal adherence at 32 weeks (OR 1.12, 95% CI .21, 5.84) or maternally-reported infant adherence at 32 weeks (OR 1.09, 95% CI .58, 2.04).Table 2Intervention effects on primary outcomes Infant HIV status (at 6 weeks)Infant HIV status (at 12 month)Maternal adherence (at 32 weeks)Mother-reported infant adherence (at 6 weeks) b (SE)95% CI b (SE)95% CI b (SE)95% CI b (SE)95% CIFixed effects  Intervention .22 (.79) − 1.32, 1.77 − 1.13 (3.03) − 7.09, 4.81 .12 (.84) − 1.53, 1.77 .09 (.32) − .53, .74 Controls (at baseline)  Age − .01 (.08) − .16, .14 − .26 (.13) − .52, .002 .02 (.04) − .07, .11 .00 (.03) − .06, .06  Education levels .18 (.27) − .34, .72 − .10 (.32) − .73, .53 .32 (.18) − .05, .69 .11 (.08) − .05, .27 Number of children .39 (.45) − .48, 1.27 2.43* (1.08) .31, .45 − .35 (.29) − .93, .22 − .01 (.15) − .31, .29  Income (monthly) − .001 (.003) − .008, .005 − .002 (.005) − .01, .007 − .001 (.001) − .003, .001 .00 (.001) − .001, .001  Diagnosis with HIV during the pregnancy .31 (.81) − 1.29, 1.93 .06 (1.20) − 2.29, 2.42 − 1.43* (.59) − 2.58, − .28 .20 (.26) − .31, .72   Children HIV positive − .17 (.85) − 1.81, 1.50 1.58 (.93) − .25, 3.42 .04 (.40) − .75, .84 − .23 (.26) − .74, .28   Partner HIV positive .21 (1.01) − 1.81, 2.11 .48 (1.39) − 2.24, 3.21 − .17 (.57) − 1.31, .95 .28 (.32) − .35, .92   Disclosure to partner − .99 (.90) − 2.68, .83 .57 (1.17) − 1.71, 2.87 .77 (.69) − .58, 2.13 .43 (.27) − .10, .95   Marital status (vs. currently married)    Not married, and not living together − .13 (1.32) − 2.81, 2.48 2.44 (2.16) − 1.79, 6.69 .25 (.58) − .88, 1.40 − .09 (.35) − .79, .59    Not married, but living together 1.07 (1.29) − 1.44, 3.65 − 3.82 (6.52) − 16.61, 8.95 .64 (.67) − .65, 1.95 − .12 (.41) − .92, .70 Random effects  Intercept .000 (.001) − .30, .31 10.54 (16.30) − 21.51, 42.61 1.66 (1.14) − .56, 3.91 .06 (.12) − .17, .30  Intervention .18 (.83) − 1.78, 1.82 1.99 (25.03) − 47.18, 51.82 .05 (.94) − 1.50, 1.62 .07 (.18) − .28, .42 Model fit  − 2LL (deviance) 83.270 84.912 333.908 588.446  Numbers of parameters 14 14 14 14  AIC/BIC 111.270/171.404 171.404/173.047 361.907/422.041 616.445/676.579  ICC (without covariates) .00 .018 .022 .098 Unstandardized logistic coefficients were shown. SE Standard error, CI confidence interval, AIC akaike information criteria, BIC Bayesian information criteria, Covariances between random intercept and random slope were fixed to 0. ICC Intra-class correlations *p < .05 BODY.SECONDARY OUTCOMES: Results for secondary outcomes are presented in Table 3. After controlling for covariates, intervention condition did not predict HIV knowledge trajectories (b = − .02, 95% CI − .04, .02, effect size = .20) or PMTCT knowledge trajectories (b = − .006, 95% CI − .02, .008, effect size = .18).Table 3Intervention effects on secondary outcomes trajectories HIV knowledgePMTCT knowledge b (SE)95% CI b (SE)95% CIFixed effects  Baseline intercept 12.36*** (.78) 10.83, 13.88 4.85*** (.48) 3.92, 5.81  Linear slope trajectory .00 (.05) − .10, .11 .000 (.026) − .05, .05  Intervention − .02 (.01) − .04, .02 − .006 (.007) − .02, .008 Controls (at baseline)  Age .000 (.001) − .003, .003 .000 (.001) − .001, .002  Education levels .001 (.005) − .008, .011 .001 (.002) − .004, .005  Numbers of children .016 (.011) − .004, .037 .005 (.005) − .005, .014  Income (monthly) .000 (.000) .000, .000 .000 (.000) .000, .000  Diagnosis with HIV during the pregnancy .022 (.017) − .010, .055 .015 (.008) .000, .030  Children HIV positive .001 (.015) − .028, .029 − .003 (.008) − .019, .012  Partner HIV positive − .004 (.016) − .035, .026 .006 (.009) − .011, .024  Disclosure to partner − .025 (.021) − .066, .016 − .003 (.009) − .020, .013  Marital status (vs. currently married)   Not married, and not living together − .002 (.020) − .040, .037 − .001 (.011) − .022, .019   Not married, but living together − .011 (.021) − .051, .030 − .016 (.011) − .039, .006 Random effects  1st Level (intra-individual variance)   Residual 4.58*** (.336) 3.92, 5.24 1.43*** (.08) 1.31, 1.63  2nd Level (inter-individual variance)   Baseline intercept 3.96*** (.71) 2.57, 5.36 .65*** (.14) .37, .93   Linear slope trajectory .01** (.002) .003, .012 .001* (.001) .000, .002   Covariance (intercept, linear slope) − .10** (.03) − .163, − .031 − .02* (.007) − .033, − .004   Covariance (intercept, intervention) − .16 (.11) − .37, .06 − .08 (.05) − .174, .013  3rd Level (site-variance)   Baseline intercept .42 (.47) − .51, 1.34 .08 (.04) − .002, .166   Intervention .00 (.005) − .01, .01 .000 (.000) .000, .000 Model fit  − 2LL (deviance) 11162.43 8316.10  Numbers of parameters 32 32  AIC/BIC 11,226.43/11,363.87 8380.10/8517.55  Average ICCs (without covariates) .104 .082 Unstandardized coefficients were shown SE Standard error, CI confidence interval, AIC Akaike information criteria, BIC Bayesian information criteria, ICC Intra-class correlations *p < .05. **p < .01. ***p < .001 BODY.DISCUSSION: This study assessed the impact of a behavioral intervention to enhance PMTCT on mother to child HIV transmission during pregnancy and maternal adherence to ARV/ART. Results suggest that the Protect Your Family intervention delivered by lay healthcare workers did not confer any additional reduction in HIV transmission to infants or increase ART adherence or HIV/PMTCT knowledge. Intervention outcomes also appear consistent with a previous review [23] suggesting that studies relying on behavioral or psychological interventions may not influence components of the PMTCT cascade such as infant HIV testing. In both SC and EI conditions, the infant HIV incidence at 12 months was less than 3.0% (SC: 2.3%, EI: 2.7%), levels that were similar to those obtained in an earlier intervention in South Africa using peer supporters (SC: 2.5% and EI: 2.6%) [38], but better than the 3.9% cumulative MTCT incidence rates identified at 12 months as part of the national cohort study in South Africa [4]. Several elements may account for these reductions in incidence. It is likely that the 2013 onset of the Option B + programme in South Africa, in which all pregnant women living with HIV are offered life-long ART, regardless of their CD4 count, was responsible for the reduction in HIV MTCT rates to levels that were too low to detect the impact of condition. In addition, a variety of national initiatives were undertaken in 2013 to enhance PMTCT at the clinic level, many elements of which duplicated study elements, e.g., support groups, peer support, suggesting that the current intervention may not have added to the existing evolving clinic environment. Given these results, it is unclear whether the intervention as delivered by lay healthcare workers could have had an impact above that achieved in the existing clinic environment. Although this study also sought to enhance PMTCT, and all women had a primary male partner, the current study did not enroll men as participants. The differences obtained between the current study outcomes and the preceding pilot (e.g., [39]) with regard to HIV/PMTCT knowledge may highlight the role of partners, who were enrolled and participated in the pilot. Additionally, about half of the women were diagnosed with HIV during the current pregnancy, which may have made HIV disclosure and male involvement during pregnancy challenging. While more than half the women reported having disclosed their HIV status to their partners, male involvement could have an added impact on intervention outcomes, including knowledge, retention, adherence and transmission. Clinic staff should continue to explore the incorporation of men in the clinic environment, as outlined in the PMTCT protocol. BODY.STUDY LIMITATIONS: The primary aim of the study was to reduce MTCT rates from the 13% at 6 weeks originally reported in the rural clinics in 2012 to under 5%. The greatly reduced HIV incidence of less than 3% in the clinical population which followed the uptake of the new Option B+ ART policy in 2013 resulted in the study being underpowered to assess the impact of condition on infant HIV status. Additionally, study follow-up rates were lower than the original target and those previously achieved in our pilot studies, and there may have been the influence of self-selection on those women who were followed to 12 months postpartum. A variety of reasons account for attrition and low attendance; these include long distances to reach the CHC, culturally condoned migration of women during pregnancy and after child birth, and migration due to economic necessity. The substantial loss to follow up of 25.9% in the control group and 35.4% in the intervention group was quite high and may reflect the unstable nature of rural communities and possibly poor utilization of health services by participants in this province. It is also likely that the impact of the intervention was reduced due to limited session attendance and low fidelity at several (2–3) sites, an outcome that may reflect the difficulty of ensuring adherence to complex intervention elements by lay healthcare workers in remote areas. Importantly, participants were also not compensated for session attendance, and most women found economic support for transportation to the CHCs for pre- and post-natal care challenging. However, through randomization, these influences would have been equally distributed between conditions. Women who did not attend antenatal care or only attended antenatal care late were not included in this trial and could possibly have introduced an additional bias. BODY.CONCLUSION: This behavioral intervention did not achieve any additional effect on HIV infant status or maternal ART adherence, HIV and PMTCT knowledge. As noted above, the ‘secular trend’ supporting PMTCT protocols seen in the SC clinics resulted in significant reductions in MTCT in all study CHCs. This likely created a “floor” effect, whereby further reduction of MTCT could be difficult to discern.

TITLE: Randomized controlled trial demonstrates the benefit of RGTA® based matrix therapy to treat tendinopathies in racing horsesNew RGTA® based matrix therapy in regenerative medicine treats tendinopathies in racing horses A randomized controlled trial was performed on racing horses, to evaluate the efficacy of a new class of therapeutic agents in regenerative medicine—ReGeneraTing Agents® (RGTA®), to treat tendinopathies. Preliminary uncontrolled studies on tendon healing in racing horses with RGTA® (OTR4131)—Equitend® showed encouraging results, justifying performing a randomized, controlled, multicenter study with a two-year racing performance follow up. The objective of this study was to evaluate the effect of Equitend® versus placebo on acute superficial digital flexor tendonitis in racing French Standardbred Trotters (ST). Twenty-two ST were randomly and blindly assigned to receive with a ratio of 2 to 1, a single Equitend® (n = 14) or placebo (n = 8) intralesional injection under ultrasonographic guidance. Horses were evaluated over 4 months, by clinical and ultrasonographic evaluations (day 0, months 1, 2, 4), and their racing performances followed up over the 2 years after treatment. During the first month of treatment, a significant decrease in the cross-sectional area (CSA) was found in the Equitend® group (p = 0.04). After 4 months, the number of Equitend® treated horses with an improved CSA was significantly higher than the placebo-treated horses (p = 0.03571). The Equitend® group returned to their pre-injury performance level, racing in, and winning, significantly more races than the placebo group (p = 0.01399 and 0.0421, respectively). Furthermore, recurrence was significantly higher in the placebo group than in the Equitend® group (71.4% vs 16.6%, p = 0.02442). In conclusion, we measured a significant, short-term, reduction effect on CSA and demonstrated a long-term beneficial effect of intralesional injection of Equitend® for the treatment of superficial digital flexor tendonitis on racing ST, racing 2. 3 times more often than placebo, with 3.3 times fewer recurrences maintaining pre-injury performance level. This study may open the way for the development of a human treatment of tendonitis. BODY.INTRODUCTION: Tendon injury remains a frequent condition in sport and race horses [1, 2], responsible for substantial wastage in the equine sports industry [3, 4], and justifying continuous development of research into new treatments. In the last decade, studies substantially focused on “biological therapies”, such as glycosaminoglycan polysulphate [5, 6], hyaluronic acid (HA) [5, 7], platelet rich plasma (PRP) [8–10], insulin-like growth factor-I [11], several sources of stems cells [12–17]. ReGeneraTing Agents (RGTA®) used in the present study belong to these “biological” or “nature mimicking” therapies. RGTA® are nano-polysaccharides engineered by substituting dextran side chains with sulfate and carboxymethyl groups, to mimic heparan sulfates (HS). HS are a subset of glycosaminoglycans (GAGs), a key element in the extracellular matrix architecture, bridging and protecting matrix proteins, as well as storing, protecting and positioning heparan binding cytokines, chemokines and growth factors. Efficacy of RGTA® on tissue repair was previously demonstrated in several experimental animal models and in human medicine in other tissues (bone, muscle, cornea, etc) [18–21]. These results triggered our interest to investigate the use of RGTA® to treat horses’ tendinitis. HS are degraded in injured tissue, due to inflammation mediated enzymatic degradation. The injected RGTA® are resistant to enzymatic degradation, and replace HS at heparan-binding sites in the matrix, allowing the extracellular matrix scaffold and growth factors to reposition in the cellular microenvironment [18]. Equitend® is an injectable formulation of RGTA® OTR4131, which has been adapted specifically for the treatment of tendon injuries, by the addition of supplementary acetate groups which alters hydrophobicity [21]. Results of a previous preliminary clinical trial on 51 horses with superficial digital flexor tendonitis, treated with a single injection of Equitend® [22] indicated an improvement of ultrasonographic appearance of tendinous lesion over the first 4 months and returned to racing 2.4 months earlier with higher level of earning (75% versus 15% in the matched controlled group). These encouraging results prompted us to design a double blind randomized, controlled multicenter clinical trial. The objective of this clinical study was to evaluate the efficacy of Equitend® (in terms of ultrasound and performance data) on superficial digital flexor (SDF) tendonitis in French Standardbred Trotters (ST) after a single intralesional injection of Equitend® or placebo. The specific objectives were: i/ to compare the short term ultrasonographic healing of SDF tendonitis between groups receiving Equitend® or placebo; ii/ to compare the racing performances of both groups with a follow-up period of two years after lesion. BODY.MATERIALS AND METHODS: BODY.TRIAL DESIGN: A multicentric prospective controlled clinical trial was conducted at the Center of Imaging and Research on Equine Locomotor Injuries (CIRALE, Normandy, France) and in 2 equine practices in Normandy (Clinique Equine du Livet, 14 140 St Michel de Livet;- Clinique Equine de Méheudin, 61150 Echouché). Institutional Animal Care and Use Committee of the National Veterinary School of Alfort (Ecole Nationale Vétérinaire d’Alfort, Maisons-Alfort, France) approved the study protocol. Inclusion criteria for client-owned racing French Standardbred Trotters (ST) were clinical and ultrasonographic signs of unilateral, acute or subacute (less than one-mon duration) SDF tendonitis. Horses were only included if the client agreed to the study design and tendons had not received intralesional injections before. Concurrent lesion which could be considered as a predisposing factor of re-injury of the SDF tendon (i.e. lameness of the opposite limb), and bilateral lesions were exclusion criteria (S1 Fig and S1 Table). Equitend® treatment and placebo solutions were randomly assigned with a ratio of 2 treatments for one placebo (this ratio was chosen to improve client compliance and facilitate recruitment). Randomization procedure and blinding are described in S1 Fig. Briefly, Excel function (RANDBETWEEN(Bottom, Top)) was used to randomize the treatments between the two groups. Blinding was in place throughout the trial and revealed only once all data, including racing performances, were collected. None of the clinicians, experimenters, owners and trainers were aware of the content of the vials at any time. BODY.CLINICAL EXAMINATION: Clinical examination included physical and dynamic evaluation. Thickening and heat of the metacarpal tendon area were evaluated as well as sensitivity to pressure of the SDF tendon on flexed limb, using a four-grade scale: 0 = no reaction at pressure, 1 = reaction to moderate pressure, 2 = reaction to mild pressure, 3 = violent reaction to mild pressure. Dynamic evaluation was performed on straight line at trot and the grade of lameness was established according to the American Association of Equine Practitioners scoring system [23]. BODY.ULTRASONOGRAPHIC EXAMINATION: All horses underwent a complete ultrasonographic examination of both metacarpal tendon areas, using a high-resolution machine equipped with 7.5 MHz linear probe coupled with stand-off pad. Severity of the SDFT injury and treatment-associated changes were assessed by measuring the cross-sectional area (CSA) of the tendon at the maximal injured site (MIS), echogenicity, transverse lesion extent, transverse lesion architecture using the angle contrast ultrasound technique (ACUST) [24], longitudinal lesion extent and architecture according to the grading system presented in S2 Table. All the ultrasound images of each horse were scored blindly by four experienced veterinarians (JMD, AGD, SJ, VC) at once, at the end of the follow-up period. Time was not randomized, and images were reviewed and scored chronologically. The scores for all horses were made at the same time by each reviewer. BODY.EQUITEND:®/Placebo treatment Equitend® product contained the GAG mimetic [OTR4131], obtained from OTR3 Inc. (Paris, France). OTR4131 is an alpha 1–6 polyglucose polymer substituted with carboxyl, sulphate and acetate groups as shown in Fig 1 [25], and prepared in 0.9% sodium chloride injectable form (0.15M NaCl), at the concentration of 10μg/mL Placebo vials were 0.9% sodium chloride only and absolutely identical to the Equitend® vials. 10.1371/journal.pone.0191796.g001Fig 1BODY.CHEMICAL STRUCTURE OF EQUITEND:® active substance OTR4131. OTR4131 is an alpha 1–6 carboxymethyl sulfate acetate glucose polymer with a molecular weight ranged between 100 to 150 kDa. After aseptic preparation of the tendon area, injection of 1ml of Equitend® or placebo was performed on flexed limb, under ultrasound guidance at the level of the MIS, using a 0.5x16 mm needle (S1 Table) as previously described [22]. Horses were sedated by intravenous administration of detomidine (8 μg/kg) and butorphanol tartrate (16 μg/kg). Following injection, a light bandage was applied. BODY.SHORT-TERM FOLLOW-UP AND REHABILITATION PROGRAM: Clinical and ultrasonographic follow-up were performed at one (M1), two (M2) and four (M4) months after treatment or placebo injection using the same criteria as on inclusion day (S3 Table). S4 Table). This program was adjusted in accordance with the clinical and ultrasonographic findings and the expectations of the owner and trainer. The use of a light shoe with wider toe and beveled branches for the front feet were recommended in all horses. BODY.PERFORMANCE FOLLOW-UP: All horses were followed up for two years after injury. The criteria used were: time to race post injury (established as the number of months from the treatment date to the first post-injury race), the number of races, the number of places, victories, the earnings per start post-injury. The results of performances after treatment were compared with results the year before injury. Racing data was obtained through websites (http://www.letrot.com/fr/fiche-horse and http://www.turf-fr.com/pack-pro). Re-injuries or end of careers were also recorded based on the information that the owners or the treating veterinarians gave us directly during the consultation or by phone call. BODY.STATISTICAL ANALYSIS: All calculations were performed using R v.3.3.2 software [26]. S4 Table summarized the collected data at the corresponding time, and their format such as continuous, binary or ordinal variables. Because the variables did not follow a Gaussian distribution, we used non-parametrical tests to analyze the data. Using a non-parametric test avoids making any assumptions about the distribution of the variables (i.e. Gaussian distribution), the independency of the variables and the minimum number of observations required to perform the test such as in parametrical test. Therefore, Kruskal -Wallis test was used to analyze non-Gaussian variables and as a non-parametric method for testing whether groups originate from the same distribution, it was used for comparing two or more independent groups of equal or different sample sizes. For the following tests, type I error-set is 5% (α = 5%). BODY.ANALYSIS OF INITIAL DATA: Baseline data at inclusion (D0, day of treatment) were described and compared between the treatment and placebo groups, using non-parametric Kruskal-Wallis test for continuous variables, chi-squared (χ2) test for binary variables, and Cochrane-Armitage test for ordinal variables. Chi-squared (χ2) test is the appropriate test to compare the distribution (i.e. frequency or proportion) between two or more groups such as recurrence. Finally, Cochrane-Armitage test is an extension of the chi-squared (χ2) test for the particular case when the categorical variable is ordinate (i.e. ordinal variable such as a grade from 0 to 4 as in ultra-sonographic variables) [27]. The test can be found in”coin” package in R-software [28]. BODY.ANALYSIS OF CLINICAL AND ULTRASOUND DATA: In order to compare the clinical and ultrasonographic outcomes over time, the difference between data at the time-point considered (M1, M2 or M4) and baseline (M0 at D0) was calculated. Linear regression models were built in which the difference between Mi (i = 1, 2 or 4) and M0 (value at inclusion) of clinical and ultrasonographic variables were the dependent variables, and independent variables were treatment/placebo group, age at the individual level and the center at group-level. It provided an estimate of the difference between the two groups because of treatment (or treatment effect) for each time-period (M0 to M1, M0 to M2, and M0 to M4), adjusted on the horse age and inclusion centers (considered as fixed effects). A generalized estimating equations (GEE) model was used for comparison of the cross-sectional area (CSA) between groups. To be closest to the normality, CSA was transformed in 1/(CSA)2. Horse age and center effect were introduced into the model as fixed-effect covariates [29]. Briefly, GEE is part of marginal models that do not require distributional assumptions for observations, only a regression model for the mean response. Marginal models provide a unified method for analyzing diverse types of longitudinal responses (i.e. continuous and ordinal variables), which avoids making assumptions about the distribution of the vector of responses; the method relies solely on assumptions about the mean response and the comparison of the coefficients and the variances [30]. BODY.ANALYSIS OF THE PERFORMANCES VARIABLES: The performances such as earnings per race, number of races and number of victories before tendinitis (one year) and after treatment (i.e. 24-months after) were analyzed by using Kruskal-Wallis non-parametric chi-squared test. The recovery of the performances before tendinitis and after treatment was analyzed using the Wilcoxon signed rank pairwise non-parametric test with continuity correction. The recurrence of tendinopathy over 2-years follow-up in both placebo and Equitend®-treated groups was assessed by Kaplan-Meier “survival” curve (“survival” R-package) and analyzed using Fleming-Harrington test [31, 32] because the effect of the therapy is more pronounced towards the middle and the end of the follow-up period (“survMisc” and “FHtest” R-packages). The recurrence over the 2-years after treatment was estimated by the odds ratio and the risk ratio using the small-sample adjusted method and compared by chi-squared test (“epitools” R-package) [33]. BODY.RESULTS: BODY.STUDY POPULATION: Between May 2008 and February 2012, forty-two (n = 42) horses met the eligibility criteria, but only twenty-four (n = 24) ST presenting with acute (or subacute) SDF tendonitis in one front limb were included in the study because of lack of owners’ compliance for the eighteen (n = 18) horses not included (S1 Fig). Two horses were excluded during the follow-up period because they presented major colic signs (S1 Fig) that required administration of non-steroidal anti-inflammatory drugs. In the final 22 horses studied, there were 3 females, 8 males and 11 geldings. The mean age was 5.4 +/- 1.4 years old (range, 3 to 8 years). Horses were randomly assigned into Equitend® (n = 14) or saline (n = 8) treatment groups and no significant differences in age, genders, duration of SDFT tendinopathies, affected forelimb, centers of investigation, lameness grades and sensitivity grades were observed between treatment groups (Table 1 and S1 Table). Hence all conditions were met for further investigation of the Equitend® treatment. 10.1371/journal.pone.0191796.t001Table 1BODY.CHARACTERISTICS OF THE STUDY POPULATION AT THE DATE OF INCLUSION.: Placebon = 8Equitend® Treatment(1 mL, OTR4131, 10 μg/mL)n = 14p-valueAge (years, mean ± SEM) 5.3 ± 0.6 5.3 ± 0.4 0.09‡ Gender (F/G/M) 1/5/2 2/6/6 0.7† Duration of SDFT tendinopathy before examination (days, mean ± SEM) 11.9 ± 2.9 25.8 ± 13.1 0.5‡ Affected forelimb R or L (n) 2/6 5/9 0.9† Centers of investigation (C/CL/CM) 5/3/0 7/6/1 0.7† Lameness grades n (%) Grade 0: 2 (25) Grade 0: 3 (21) 0.8+ Grade 0.5: 0 (0) Grade 0.5: 2 (14) Grade 1: 4 (50) Grade 1: 5 (36) Grade 2: 2 (25) Grade 2: 4 (29) Sensitivity grades n (%) Grade 1: 3 (38) Grade 1: 1 (7) 0.1+ Grade 2: 2 (25) Grade 2: 5 (36) Grade 3: 3 (38) Grade 3: 8 (57) Horses were randomly assigned into either Equitend® treatment or placebo group. All horses included in this study are Standardbred trotter (ST). Comparison between placebo and Equitend® by using ‡ unpaired t-test, † Chi-squared test with Yates’ correction, and + Cochrane-Armitage test for ordinal variables. BODY.SAFETY: As expected from preclinical safety and previous studies [22], intralesional injection was well tolerated for all horses and no adverse local or systemic effects were reported after treatment. BODY.CLINICAL AND ULTRASOUND FOLLOW-UP: Analysis of clinical parameters on the day of inclusion (day 0) into the study revealed no significant difference between Equitend® and saline treated groups. Ultrasonographic examination revealed an unexpected increase in the transversal lesion extent (p = 0.04) and a higher CSA at MIS (p = 0.01) in the Equitend® treated group at day 0 (Table 2). 10.1371/journal.pone.0191796.t002Table 2BODY.ULTRASONOGRAPHIC AND PERFORMANCE DATA AT D0, AND COMPARISONS ACCORDING TO TREATMENT GROUPS.: VariablesGradeOverall population (n = 22)Control group (n = 8)Treatment group (n = 14)p-valueUltrasonographic variables Echogenicity 2 3 (14%) 1 (12.5%) 2 (14%) 0.7 3 12 (55%) 4 (50%) 8 (57%) 4 7 (32%) 3 (37.5%) 4 (29%) Transversal architecture 2 1 (5%) 0 (0%) 1 (7%) 0.08 3 8 (36%) 3 (37.5%) 5 (36%) 4 13 (59%) 5 (62.5%) 8 (57%) Longitudinal architecture 2 2 (9%) 1 (12.5%) 1 (7%) 0.7 3 14 (64%) 5 (62.5%) 9 (64%) 4 6 (27%) 2 (25%) 4 (29%) Trans. Lesion extent 1 2 (9%) 2 (25%) 0 0.04 2 5 (23%) 3 (37.5%) 2 (14%) 2.5 2 (9%) 0 (0%° 2 (14%) 3 8 (36%) 2 (25%) 6 (43%) 4 5 (23%) 1 (12.5%) 4 (29%) Long. Lesion extent 3 2 (9%) 2 (25%) 0 0.3 3.5 1 (5%) 0 (0%° 1 (7%) 4 19 (86%) 6 (75%) 13 (93%) Cross Section Area—tendonitis side 1.55 (1.27–1.77) 1.57 (0.37) 1.26 (1.14–1.45) 1.33 (0.32) 1.66 (1.13–1.88) 1.70 (0.34) 0.01 Cross Section Area—contralateral limb 0.85 (0.78–0.94) 0.88 (0.16) 0.79 (0.76–0.86) 0.80 (0.07) 0.87 (0.81–1.10) 0.93 (0.18) 0.08 Values are expressed as median (inter-quartile interval) and mean (standard deviation) for continuous variable, and as number (percent) for categorical variables. While the CSA did not significantly change in the placebo treated group (p = 0.2), there was a significant decrease in CSA from M0 to M1 (from 1.70 to 1.61 cm2) in Equitend® treated horses (p = 0.04, Tables 3 and 4) whereas in the placebo group CSA remained constant (from 1.33 to 1.34 cm2). These differences could be explained because in Equitend®-treated horses CSA was significantly higher compared to placebo group at the time of inclusion (M0) (p = 0.01). 10.1371/journal.pone.0191796.t003Table 3BODY.TREATMENT EFFECT ON CROSS SECTION AREA (CSA) AT THE MAXIMAL INJURED SITE (MIS) ON THE AFFECTED LIMB IN THE PERIOD M:0 to M1—General Estimating Equations (GEE) model. VariablesCoefficient (95% CI)p-valueChange of CSA in the control group between M0 and M1 -0.03 (-0.09 to 0.02) 0.2 Difference between the two groups at baseline -M0 -0.18 (-0.28 to -0.76) 0.01 Treatment effect on CSA between M0 and M1 0.07 (0.002 to 0.14) 0.04 CI: confidence interval. 10.1371/journal.pone.0191796.t004Table 4BODY.MEAN CSA MEASURES BEFORE AND DURING THE FIRST FOUR MONTHS OF TREATMENT.: CSA mean ± SD cm2M0M1M2M4Placebo (n = 8) 1.33 ±0.32 1.34 ± 0.16 1.29 ± 0.14 1.33 ± 0.17 Equitend (n = 14) 1.70 ± 0.34 1.61 ± 0.34 1.60 ± 0.36 1.63 ± 0.35 Interestingly, the number of Equitend®-treated horses (n = 7) for which the CSA decreased more than 10%, after 4-months of treatment, was significantly higher (p = 0.03571) compared to the number of placebo-treated horses (n = 1, Fig 2). The average CSA improvement in Equitend®-treated horses (n = 7) was about 17% during this 4-months of treatment, with a CSA improvement in these 7 horses (Fig 2) ranged from an average of 1.80 cm2 (M0) to 1.50 cm2 (M4). In both groups, CSA did not change for 5 horses and deteriorated for 2 horses. 10.1371/journal.pone.0191796.g002Fig 2BODY.IMPROVEMENT OF CSA AFTER 4 MONTHS OF EQUITEND:® treatment. Graphics represent CSA (cm2) at the time of inclusion (M0) and after 4-months (M4) treatment for each horse in placebo group (left panel) and Equitend®-treated group (right panel). The table represent the number of horses in each treated group that improved more than 10% the CSA (■), not improved (■ Decrease or increase of CSA less than 10%), or worsened more than 10% (■). Data were compared by asymptotic Cochran-Armitage test. BODY.RACING DATA ANALYSIS: Among the 22 included horses, 19 horses returned racing: 7 out of 8 horses (87.5%) in the placebo group and 12 out of 14 horses (85,7%) in the Equitend®-treated group. The three horses (one in placebo group and 2 in treated group) which did not resume racing, were retired for reasons unrelated to their tendonitis. The proportion between groups was not dissimilar (p = 0.974, chi-squared test). Analysis of performance in the year prior to the tendinopathies, such as number of races, number of victories and earnings per race did not show any differences between placebo and Equitend®-treated groups. Both groups are therefore considered as even. Equitend®-treated horses raced significantly more than the placebo group (p = 0.01399) and the number of victories was significant higher (p = 0.0421, Table 5). Moreover, Equitend®-treated horses returned to their pre-injury performance level whereas placebo-treated horses did not (Table 5). In the placebo group the median number of races before treatment was 14 (interquartile [10–16]) whereas 24 months after injection it was 6 (interquartile [4.5–9.5], p = 0.0446). In the Equitend®-treated group, values before treatment were 15 [interquartile: 7.5–25] and became 15.5 after 24 months of treatment (interquartile [10–22.5], p = 0.6382). 10.1371/journal.pone.0191796.t005Table 5BODY.EQUITEND:®-treated horses have better performances and recovery compared to placebo a year before tendinitis and over the two years after inclusion. 12 months before treatment24 months after treatmentRecovery (Before vs After)Performances of the horsesPlacebo group n = 7Treatment group n = 12*p-valuePlacebo group n = 7Treatment group n = 12*p-valuePlacebo #p-valueTreatment #p-valueEarnings/race median [interquartile] 2845 [1484–6259] 2636 [1486–3090] 0.5617 439 [91–3131] 1296 [649–3302] 0.3525 0.1094 0.2593 Number of races median [interquartile] 14 [10–16] 15 [7.5–24.25] 0.5243 6.0 [4.5–9.5] 15.5 [10–22.5] 0.01399 0.0446 0.6382 Number of victories median [interquartile] 1 [1–2.5] 2 [0.75–3.5] 0.8293 0 [0–0] 1.5 [0–2] 0.0421 0.0170 0.2647 Values are expressed as median [inter-quartile range]. *p-values were calculated using the Kruskal-Wallis non-parametric chi-squared test. #p-values were calculated using the Wilcoxon signed rank pairwise non-parametric test with continuity correction. Similar results were observed for the number of victories. In the placebo group, no victories were reported after treatment, while the Equitend®-treated horses performed as before injury (Table 5). The recurrence of tendinopathy over 24-months follow-up was assessed by Kaplan-Meier curve and showed that the placebo-treated group had significantly more recurrence of tendinopathy compared to Equitend®-treated group (Fig 3® group compared to the placebo group (Table 6). 10.1371/journal.pone.0191796.g003Fig 3BODY.EQUITEND:®-treated horses had significantly fewer recurrences of tendinopathy over 24-months follow-up. Kaplan-Meier curve shows recurrences of tendinopathy over 24-months follow-up of placebo (n = 7) and Equitend®-treated (n = 12) groups. p-value was estimated using Fleming-Harrington weights, emphasizing differences later in time. Fleming-Harrington is a Kaplan-Meier (product-limit) estimator. 10.1371/journal.pone.0191796.t006Table 6BODY.PLACEBO GROUP HAS A HIGHER RISK OF RECURRENCE THAN EQUITEND:®-treated group over the two years after inclusion. RecurrencePlacebo groupn = 7Treatment groupn = 12 Yes 4 2  No 3 10 Odds ratio 3.333 1 Risk ratio 2.476 1 #p-value 0.0671 The odds ratio and the risk ratio are estimated by the small-sample adjusted method and # compared by Chi-square test BODY.DISCUSSION: This study confirms efficacy of RGTA® based matrix therapy in the treatment of tendinopathies. Sport and race horses are considered as a relevant model for humans over use tendonitis such as those developed in repeated motions and intense sport practice [34]. Therefore, the results presented here are expected to be supportive for a human efficacy study, although such a study needs to be designed and adapted. The reduced recurrence rate measured in this study is also a promising indicator to follow for human tendonitis. Another parameter associated with RGTA® not reported in this study is pain relief observed in cornea [19, 35] and skin lesion [36]. For a human study, the pain reduction parameter would be of great interest. Double blind, randomized, controlled, prospective clinical study is unanimously recognized as the gold standard for a clinical trial [37], however it is hard to recruit racing horses to reach a sufficient population to avoid any bias. This limit is due to the lack of owner compliance and represents a weakness of this study since the statistical analysis was limited by the small population. To our knowledge, we identified two field randomized clinical trials showing some efficacy of autologous MSC cells [13, 15]. Dyson (2004) was not able to perform a randomized placebo controlled trial because of this inherent client limitation [5]. To counteract these small groups a very homogeneous population with a detailed and long-term follow-up was studied. Another limit of the trial was the difference of lesion severity between the 2 groups at the time of inclusion, the CSA being significantly higher in the Equitend® –treated group compared to the placebo group. This difference could not have been expected nor prevented as the randomization was blinded. Indeed, CSA is the most objective measure of tendon pathology and can be assessed accurately using ultrasound for the tendinopathy follow-up, as extensively described before [38–40] but not necessarily directly related to the extent of the pathology. Marr et al. (1993) identified that the severity of the lesions was related to the outcome on a population of 73 National Hunt racehorses [41]. It is reasonable to assume that difference between the 2 groups could have been more demonstrative in favor of the Equitend® group if initial lesions were similar. Most previous published data referred to retrospective [11], uncontrolled [13, 15, 16, 42] or matched [5, 9, 10, 22] trials. A study published by Smith et al. (2013), described a randomized and controlled clinical trial, to estimate the efficacy of autologous bone marrow stem cells on naturally occurring tendinopathy, but no performance follow-up was presented [43]. As the population studied was exclusively composed of retired National Hunt horses, the conclusion was based only on ultrasonographic healing, and results cannot be extrapolated to racehorses in activity. A recent prospective controlled clinical trial evaluated the effect of intralesional PRP treatment on naturally occurring SDF tendonitis [44]. The authors demonstrated that a higher proportion of treated horses were able to reach their previous or higher level of performance compared with placebo treated horses (80 versus 50% at 12 months, 60 versus 50% at 24 months). Nevertheless, the population studied was not homogeneous and only composed of sport and pleasure horses of various ages and levels of activity. To our knowledge the present study is the first prospective controlled clinical trial on racehorses with 2 years follow-up. According to the first Havemeyer workshop on equine tendon disease [37], evaluation of the efficacy of a treatment requires analysis of several objective outcome parameters (i.e. re-injury rate, proportion of horses racing more than 3 or 5 times). Most clinical trials previously published were performed on flat and National Hunt racehorses or sport horses [2, 5, 10, 11, 16, 41, 43]. Few were made on Standardbreds [9, 11, 42, 45], and only three considered superficial digital flexor tendonitis [15, 42, 45]. The choice of ST in this study was based on the large population available in France, the easy accessibility to performance data, and the simple management of racing career (ST resume racing as soon as possible and can race all year long). As described by Cheetham et al. (2010), several factors as age, sex, breed, discipline, gait, track surface (for Thoroughbred), could interfere with outcome [46]. In the present study, these parameters were very homogeneous between both studied groups. No difference of breed, sex and age, was identified, and trot is the only gait used in France. Furthermore, the placebo and Equitend®-treated groups also matched duration of SDFT tendinopathies, affected forelimb, centers of investigation, lameness grades and sensitivity grades. Interestingly, echography analysis before treatment indicated a significant difference between the two groups. The Equitend® group showed higher deterioration of the transectionnal lesion extent and cross-sectional area. After one month of treatment, no improvement was measured in the placebo group while the Equitend®-treated group showed significant improvement for both parameters. CSA was significantly improved by 17% at M4 in 7 Equitend® treated horses while only 1 horse in the placebo group spontaneously improved (p = 0.03571), suggesting that this improvement might have been even higher at 6 months as reported in other studies [16, 43, 47]. To ensure homogeneous conditions, the same rehabilitation program and shoeing were recommended for all horses. Activity was adapted according to the findings at ultrasound and clinical examinations as described above in “Short-term follow-up and Rehabilitation program” and S3 Table. A synthesis of the previously reported rehabilitation protocols was built on the 2007 Havemeyer symposium [37], but was dedicated to Thoroughbred (TB) racehorses or sport horses. Interestingly, although recovery under our treatment is faster, performance data registered were similar than those reported on TB [5, 10, 11, 13]. It would suggest that recovery period for SDF tendonitis on ST could be reduced compared to TB racehorses. Emphasis was put on long-term analysis, with numerous performance data studied and up to 2 years follow-up after injury. Racing performances of the two populations were different: out of 8 Placebo treated horse 7 went back to racing but ran fewer times than previously whereas the Equitend® group (12 out 14 horses) resumed similar activity and performances as before injury, running significantly (2.3 times) more than the placebo group. An average of four times higher recurrence was noted in the placebo group compared to Equitend® treated group. Dyson (2004) reported re-injury rates in various intralesional treatment groups with 42.5% for HA, 44.4% for PSGAG treatment or 45.5% (when including contralateral limb) for β-aminopropionitrile fumarate. Published data on the use of bone marrow mesenchymal stem cells showed lower re-injury rate with respectively 32.7% (when including contralateral limb) in Godwin et al. (2013) study [37, 43], and only 13% in Smith et al. (2013) study [37, 43]. In this last study, the re-injury rate was the unique outcome parameter studied, and analysis of several performance criteria is recommended to evaluate a treatment on tendonitis [37, 43]. In a study comparing performance of TB affected with SDF tendonitis matched with sound horses, O’Meara et al. showed that the assessment of the outcome of horses with SDF tendonitis using the number of races post injury required a minimum of 5 races post injury to be a useful indicator [4]. In the present study, a higher number of races post injury in the Equitend® group was noted, with a higher rate of horses who competed more than 5 times. With respectively 78,6% and 64,3% of horses who competed more than 5 and 10 times on the Equitend® group, these results compare favorably with our previous analysis for the same category of horses (Standardbreds) [42]. Hogan et al. (1995) published data on horses with desmotomy of the accessory ligament of the superficial digital flexor tendon (AL-SDFT), with a rate of 69% of horses racing more than 5 times [42]. Sawdon (1996) showed that 52% of horses competed more than 5 times with only conservative management [45]. In a more recent study, with use of autologous bone marrow aspirate alone or combined with desmotomy of the AL-SDFT, Russel et al. (2016) observed that 62% of treated horses raced more than 5 times and 53% more than 10 times [15]. Contrary to other treatments such as PRP, bone marrow or stem cells, Equitend® is a stable, synthetic molecule. Moreover, only a single intralesional injection with a small volume of product was performed in this study. Therefore, Equitend® could be considered by practitioners, as a more practical therapeutic option than PRP or stem cells. More extensive studies should address the use of Equitend® at earlier stage after tendinopathies injury as suggested by the mode of action of this molecule adapted to acute injury and to evaluate the benefit of a second injection in case of inconclusive ultrasonographic follow up data. BODY.CONCLUSION: Results of this study combined with the previous preliminary trial performed on Equitend® demonstrate that a single intralesional injection of Equitend® is safe and has beneficial effect for management of tendonitis in horses. Equitend®-treated horses recovered better then placebo treated horses, returning to a performance level similar to their pre-injury level. Equitend is the first RGTA® product developed to be injected for clinical uses and for a veterinary drug application, although many experimental tissue lesion animal models have demonstrated efficacy by injection. The results presented in this study will support development for a human use. BODY.SUPPORTING INFORMATION: S1 FigBODY.FLOW DIAGRAM OF EQUITEND:® clinical trial. The figure summarized the steps followed to include the 24 horses on the clinical trial based on the consort recommendations (http://www.consort-statement.org/consort-statement/flow-diagram). (PDF) Click here for additional data file. S1 TableBODY.DESCRIPTION AND CLINICAL HISTORY AND TREATMENT OF EACH HORSE.: Horses were randomly assigned into either Equitend® treatment or placebo groups. Horses were sedated with detomidine (8 μg/kg) and butorphanol (16 μg/kg) and injection sites were prepared by shaving and sterilization. Equitend® (1 mL, OTR4131, 10 μg/mL) or control saline solution was administered via intralesional injection under ultra-sonographic guidance into each injured tendon at the maximal injury site (MIS), using 25” gauge disposable sterile syringes. During the entire trial period, the composition of the solutions was known only by OTR3 to ensure blinding throughout the study. Following injection, a light bandage was applied. The horses were kept in stables for 24 h prior to their return to paddocks. Bandages were removed 24 h after injection and the injection site was monitored. (PDF) Click here for additional data file. S2 TableBODY.SEMI-QUALITATIVE AND QUANTITATIVE ULTRASONOGRAPHIC CRITERIA FOR CHARACTERIZING THE SDFT LESIONS.: MIS: Maximal injury site; CSA: cross-sectional area; SDFT: superficial digital flexor tendon. (PDF) Click here for additional data file. S3 TableBODY.SUMMARY OF THE CLINICAL, ULTRASONOGRAPHY AND PERFORMANCE VARIABLES, THEIR MEASUREMENT TIMES, AND FORMAT.: *All data were collected for the horse, except the CSA that was collected for both side (tendonitis one and healthy counter-side). Meaning of the time-points: D0: inclusion date, and day of injection into the forelimb of the experimental treatment or placebo. M1, M2 and M4: repeated measurements of the variables. M12 and M24: for the calculation of number of race and victory(ies) 12 months before inclusion and 24 months after inclusion respectively. (PDF) Click here for additional data file. S4 TableBODY.GUIDELINES FOR THE REHABILITATION PROGRAM ADJUSTED ACCORDING TO CLINICAL AND ULTRASONOGRAPHIC FINDINGS AT EACH CONTROL (M:1, M2, M4). (PDF) Click here for additional data file.

TITLE: Prebiotic and Probiotic Fortified Milk in Prevention of Morbidities among Children: Community-Based, Randomized, Double-Blind, Controlled TrialRole of Probiotic in Morbidity ABSTRACT.BACKGROUND: Recent reviews suggest common infectious diseases continue to be a major cause of death among preschool children in developing countries. Identification of feasible strategies to combat this disease burden is an important public health need. We evaluated the efficacy of adding prebiotic oligosaccharide and probiotic Bifidobacterium lactis HN019 to milk, in preventing diarrhea, respiratory infections and severe illnesses, in children aged 1–4 years as part of a four group study design, running two studies simultaneously. ABSTRACT.METHODS AND FINDINGS: In a community based double-masked, randomized controlled trial, children 1–3 years of age, willing to participate, were randomly allocated to receive either control milk (Co; n = 312) or the same milk fortified with 2.4 g/day of prebiotic oligosaccharide and 1.9×107 colony forming unit (c.f.u)/day of probiotic Bifidobacterium lactis HN019 (PP; n = 312). Children were followed up for 1 year providing data for 1–4 years. Biweekly household surveillance was conducted to gather information on compliance and morbidity. Both study groups were comparable at baseline; compliance to intervention was similar. Overall, there was no effect of prebiotic and probiotic on diarrhea (6% reduction, 95% Confidence Interval [CI]: −1 to 12%; p = 0.08). Incidence of dysentery episodes was reduced by 21% (95% CI: 0 to 38%; p = 0.05). Incidence of pneumonia was reduced by 24% (95% CI: 0 to 42%; p = 0.05) and severe acute lower respiratory infection (ALRI) by 35% (95% CI: 0 to 58%; p = 0.05). Compared to children in Co group, children in PP group had 16% (95% CI: 5 to 26%, p = 0.004) and 5% (95% CI: 0 to 10%; p = 0.05) reduction in days with severe illness and high fever respectively. ABSTRACT.CONCLUSIONS/SIGNIFICANCE: Milk can be a good medium for delivery of prebiotic and probiotic and resulted in significant reduction of dysentery, respiratory morbidity and febrile illness. Overall, impact of diarrhea was not significant. These findings need confirmation in other settings. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT00255385 BODY.INTRODUCTION: Globally, 5.2 million children under five years of age die every year due to preventable infectious diseases like pneumonia, diarrhea, malaria and measles [1]. Recent findings suggest 21% of global deaths and disability adjusted life years in children younger than 5 years of age are attributable to under nutrition and its synergistic relationship with preventable infectious diseases [2], [3]. Interventions are needed for prevention of these diseases for achieving Millennium Development Goals for child survival and reduction in child mortality by two-thirds by 2015 [4]–[6]. Probiotic bacteria benefit the host by adhering to the gut epithelium, stimulating host immune response, inhibiting epithelial and mucosal adherence of pathogens and producing antimicrobial substances [7]. Non-digestible carbohydrates that favor the growth and/or activity of probiotic bacteria are termed Prebiotics [8]. There has been increasing evidence in the last decade for efficacy of probiotic agents in treatment of acute diarrhea [7], [9]–[12], persistent diarrhea [13] and prevention of antibiotic associated diarrhea [14]–[16]. The evidence for impact on non-diarrheal illnesses has been unclear [17]–[20]. The data on efficacy for prevention of morbidity has been limited to small studies, mainly hospital based with short follow up or day care center based, with small sample size and short follow-ups [21]–[22]. Until date, only three randomized controlled trials have reported role of probiotics in prevention of community-acquired diarrhea [18], [20], [23]. Of these, two were in day care centers [18], [20] one of which was in developed country [20]. Data on combined use of prebiotics and probiotics in preventing common childhood illnesses in a community setting from developing countries is lacking as are data evaluating the effect on illness other than diarrhea. We undertook a community-based, doubled-masked randomized trial with four arms to evaluate the effect of two different milk interventions in comparison to their respective control groups (essentially running two trials concurrently with a common randomization). Two groups evaluated impact of fortifying a regular milk with micronutrient bundle in comparison to same milk without fortification; and the other two groups evaluated fortification of a pre-fortified premium milk with prebiotic and probiotic in comparison to same milk without prebiotic and probiotic fortification. In this paper we are reporting the results of the two arms evaluating efficacy of consumption of prebiotic oligosaccharide and probiotic Bifidobacterium lactis HN019 fortified milk for a period of one year, in preventing childhood morbidity among children 1–4 years old in a peri-urban community based setting in India. The results of the other two arms are reported separately in a companion paper [24]. BODY.METHODS: The protocol for this trial and supporting CONSORT checklist are available as supporting; see Checklist S1 and Protocol S1. BODY.POPULATION DESCRIPTION AND ELIGIBILITY: The trial was carried out between April 2002 and April 2004, at Sangam Vihar, New Delhi, India. Detailed population description has previously been reported [25]. Briefly most of the inhabitants are migrants from eastern Uttar Pradesh, Bihar and Rajasthan. Literacy rates are low with 50% of the women being illiterate. About 80% men work as daily wage laborers or in factories, while 95% of women are housewives. Average family income is below 600 $/year. Community has minimal access to sewage, drinking water and paved roads. Diarrhea and respiratory illnesses are common causes of childhood mortality and morbidity. Breastfeeding is a common universal practice in the first year of life, though it starts declining after first year. From a regularly updated database, all permanent resident families in area with children 1–3 years were invited to participate in the study and consent sought. Children with severe malnutrition needing rehabilitation by protocol were to be excluded; however, no such child was encountered. Children allergic to milk or with history of lactose intolerance were not enrolled into the trial. BODY.ETHICS: The study protocol was approved by the human research and ethical review committees at the Johns Hopkins University, USA and the Annamalai University, India. The purpose of the study was explained to the parents in the local language, and a written informed consent was obtained. Procedure consisted of supervisor visiting the household and in presence of a third party, obtaining the consent from the mother or father after reading the consent form to them. Parents were given a choice to sign the consent form or if they were illiterate and/or could not sign, supervisor and the witness signed to document the consent. This procedure had been approved by both institutional review boards' as majority of the mothers cannot sign in this population and taking thumb impression is stigmatized due to misuse during colonial era. BODY.ENROLLMENT AND BASELINE EVALUATION: Consented children were enrolled into the study and scheduled for baseline assessments in the clinic. Detailed physician examination of the child, blood sampling for assessment of body iron stores and anaemia, and weight (SECA Corporation, Columbia MD/ATCO Weighing Solutions Company Ltd, India) and height/length (Shorr Productions, Olney, MD) measurements were undertaken. Venous blood sample was collected by a trained nurse in trace free syringes to avoid micronutrient contamination. Blood samples were analyzed for a detailed hemogram using a coulter automated flow cytometer (Beckman-Coulter, Fullerton, CA), zinc protoporphyrin using hematoflourometer (AVIV Biomedical, Lakewood, NJ), serum ferritin and serum transferrin by commercial enzyme linked immunosorbant assay kits (Spectro Ferritin Kit, Spectro Transferrin kit; Ramco Laboratories, Inc, Houston, Texas). In our study, children with hemoglobin (Hb) ≤100 g/L were considered anemic and classified as iron deficient if they satisfied any two of the following four conditions: Serum ferritin ≤12 µg/L, serum transferrin >8.3 µg/ml, hematocrit ≤30%, zinc protoporphyrin ≥80 µmol per mole of heme. BODY.RANDOMIZATION AND MASKING: The study was a double blind randomized controlled trial with four arms wherein we evaluated the effect of two separate interventions in comparison to their respective controls. Four letter codes namely A, B, C or D were identified for each treatment group across the two trials. Permuted fixed block randomization with block length of 16 was used. Two separate randomization lists were generated using an in-house computerized randomization schedule - Strata one for baseline Hb>70 g/L and strata two for baseline Hb≤70 g/L. Based on their baseline Hb, children were stratified into these strata and assigned a treatment code. The supplementation sachets were identical in color, size (weight 32 g), taste, and were labeled with letter code. In the field, the letter code of the supplementation box was stripped off and labeled with child's identification information. The product corresponding with the letter code was known only to the manufacturing supervisor at Fonterra Brands (Singapore) Pte. Ltd. It was not known to investigators or anyone in the field until study completion and analysis. Morbidity impact of the micronutrient arm and its control has been previously published [25]. BODY.SAMPLE SIZE ESTIMATION: The sample size was determined on the assumption that prebiotic and probiotic intervention would decrease diarrhea incidence by 15% and episodes of pneumonia by 25% with alpha of 0.05 and 90% power. Allowing a 10% increase in sample to account for variation in rates and 10% more for possible attrition, it was decided to enroll 312 children per group. BODY.INTERVENTION: Fonterra Brands (Singapore) Pte. Ltd. provided fortified and control milk powder packed into 32 g single serve sachets. During enrollment into the study, mothers were explained the procedure to reconstitute the milk powder before feeding. Both groups received 21 sachets weekly at home by the Milk assistant (MA), with an advice to consume three sachets a day. The intervention was carried out for 1 year. Data on compliance and unused sachets were collected every week. Intervention (fortified milk per 3 serves a day) was designed to deliver 2.4 g of prebiotic oligosaccharide and 1.9×107 c.f.u of probiotic Bifidobacterium lactis HN019. Oligosaccharide acted as a substrate, to facilitate the growth and activity of Bifidobacterium lactis HN019 in the gastrointestinal tract. The composition of milk in PP and Co group is given in Table 1. Irrespective of group allocation, all children with severe anemia at baseline were given a therapeutic dose of iron for 3 months in addition to their milk supplement. 10.1371/journal.pone.0012164.t001Table 1BODY.COMPOSITION OF PREBIOTIC AND PROBIOTIC FORTIFIED MILK AND CONTROL MILK.: Nutritive Value (per day) Prebiotic & Probiotic fortified Milk (PP) Control milk (CO) Energy (kJ) 1890 1890 Protein (g) 20.1 20.1 Carbohydrates (g) 50.1 50.1 Fat (g) 19.2 19.2 Vitamin Aa (µg) 300 300 Vitamin D (µg) 5.1 5.1 Vitamin Eb (mg) 6 6 Vitamin C (mg) 48 48 Folate DFEc (µg) 114 114 Vitamin B 12(µg) 2.7 2.7 Calcium (mg) 720 720 Phosphorous (mg) 540 540 Iron (mg) 5.4 5.4 Zinc (mg) 3.3 3.3 Prebiotic-oligosaccharides (g) 2.4 0 Bifidobacterium lactis HNO19 (cfu)d 1.9×107 0 aRetinol activity equivalents. bα-tocopherol equivalents. cDietary Folate Equivalents. dColony forming unit. Milk Ingredients: Skim milk, Corn syrup solids, Cream, Sucrose, Vegetable oils (soya and sunflower), Lactose, Fish oil, Lecithin, Vanillin, Vitamins: Vitamin A, Vitamin D3, Vitamin E, Thiamin hydrochloride, Pyridoxine hydrochloride, Vitamin C, Folate, Niacinamide, Minerals: Ferrous sulphate, Zinc sulphate (Fortified milk contains additional prebiotic and Bifidobacterium lactis HNO19). BODY.FOLLOW UP OBSERVATION: A team of Morbidity supervisors (MS) undertook twice weekly home visits to collect prospective follow up morbidity information. Information on compliance to intervention was collected by both Milk (MA) and Morbidity (MS) teams. Before the start of the trial, we organized workshops to train and establish reliability among the field team members for measuring respiratory rate (RR), temperature and lower chest in drawing. Reliability exercises were repeated at scheduled intervals. At each home visit, morbidity information for each of the previous 3–4 days since last visit was recorded, including number of diarrheal stools, consistency of stools and blood in stools, pneumonia, fever, ear discharge, measles, vomiting and feeding history. During these biweekly visits, MS team measured RR and temperature of the child, and looked for signs of lower chest in-drawing. Whenever any of these parameters was found to be more than the normal range, children were referred to study physician for further examination. Household was revisited by MS/MA on the next day, in case the child or the parent was not available on a scheduled visit day. Two levels of supervision and random checking were established above the MA and MS level to ensure quality control of data. Mothers were advised to contact study physicians at the clinic if they felt that the child was sick between visits. Treatment of diarrhea, dysentery and pneumonia as per WHO guidelines was provided free to the participating children throughout the study. All visits either to the study physicians or to private physicians were recorded. The anthropometric measurements were repeated after 6 months and one year of intervention. The blood sampling was repeated after one year of supplementation. BODY.DEFINITIONS OF OUTCOMES: Primary outcomes were not explicitly prespecified in the protocol; the intent was to evaluate impact on common childhood illnesses including diarrhea, pneumonia, and febrile illness. However, the sample size was estimated based upon the effects on diarrhea and pneumonia. Diarrhea was defined as ≥3 loose or watery stools in 24 hours, and diarrheal episodes were considered recovered on first day of three diarrhea free days. Dysentery was defined as diarrhea with visible blood in stools. We used field based pneumonia definition [26]. Severe ALRI was defined as RR> = 50/min, Pneumonia was defined if a) severe ALRI was present or b) RR was> = 40/min but was accompanied by either lower chest in-drawing or temperature of > = 37.7°C. Axillary temperature of ≥38.4°C was considered as high fever. Severe illness was defined as days with temperature ≥38.4°C or hospitalization or RR≥50/min or chest in-drawing associated with it. BODY.DATA MANAGEMENT AND STATISTICAL ANALYSIS: The data collected in the field on a pre-designed data collection form was entered, collated and stored in the relational database management system developed in Oracle 8i with stringent range, consistency and logical checks. Real time data entry, data being entered by the end of next day after data collection, ensured data quality and accuracy. A double data entry and manual checking of frequencies was performed during data cleaning. We performed intent to treat analysis, i.e. all children were included in analyses irrespective of supplement adherence. For children out-migrating or withdrawing from the study, data were included until the date of censorship. Person-time analysis was performed with actual follow-up as denominator. For the effect on incidence (diarrhea, ALRI, dysentery, measles), relative risk has been estimated using Poisson regression and for prevalence, odds ratio has been estimated using General Linear Model for binomial outcomes (maximum likelihood logit estimation for grouped data). Both estimations were performed in STATA 9.2, (Stata Corp, Union Station, TX, USA), and SPSS ver. 12.0 (SPSS Inc., NJ, USA). Anthropometric Z scores were calculated using WHO standards [27]. BODY.RESULTS: Out of 651 eligible children contacted, 624 children [312 in intervention (prebiotic and probiotic milk (PP), 312 in control milk (Co)] were enrolled into the trial (Figure 1). At enrollment, children allocated to the groups were comparable for socio economic, demographic descriptors, hematology and nutritional status (Table 2). At baseline, 55% of children in both the groups were partially breastfed. The adherence to study milk feeds was high and similar in both groups, 84.0% children in the PP group and 82.7% in Co group consumed two or three servings on >80% days. This did not vary by the intervention period. No adverse event because of intervention was observed during the course of the study. Of the total follow up period, information was not available for 19% of the child-days in the prebiotic and probiotic group and 21% of the child days in the control group due to non-availability of the children and their parents. Six children in the prebiotic and probiotic group and five children in the control group had withdrawn consent during the follow up. 10.1371/journal.pone.0012164.g001Figure 1BODY.SCHEMATIC REPRESENTATION OF TRIAL DESIGN.: 10.1371/journal.pone.0012164.t002Table 2BODY.BASELINE CHARACTERISTICS OF PREBIOTIC AND PROBIOTIC FORTIFIED AND CONTROL MILK GROUPS.: PP Group (n = 312) Co Group (n = 312) Age (mo)a 22.2±6.4 22.9±6.8 (21.7)b (21.6) Age (≤24 months)c 191 (61.2) 191 (60.9) Illiterate fatherc 43 (13.8) 54 (17.3) Illiterate motherc 152 (48.7) 155 (49.7) Occupation father Daily wage laborc 105 (33.7) 112 (35.9) Occupation mother Housewivesc 299 (95.8) 301 (96.5) Socio economic status scorea 7.66±2.57 7.10±2.45 Water supply Tap waterc 189 (60.6) 195 (62.5) Hematological Status Hemoglobina , d (g/L) 91.1±15.6 91.0±14.9 93.0 (61.0;114.0)e 92.0 (63.0; 114.0) Zinc protoporphyrina , d (µmol/mole heme) 193.46±125.65 199.12±124.99 151.5 (42.0; 481.7)e 167 (55.8; 456.6) Serum transferrina , d (g/L) 15.25±8.85 15.21±8.79 12.97 (5.68; 35.70)e 13.22 (5.22; 35.49) Serum ferritina , d (µg/L) 9.23±7.96 9.87±9.09 6.81 (2.29; 25.7)e 6.75 (2.15;29.76) Redcell distribution widtha , d (%) 19.36±2.75 19.35±2.68 19.1 (15.1; 24.1)e 19.3 (14.9; 23.8) Iron deficient anemicc 158 (54.1) 168 (56.9) Nutritional status Normalc 107 (34.3) 95 (30.4) Wasted and Stuntedc 53 (17.0) 46 (14.7) Wastedc 15 (4.8) 14 (4.5) Stuntedc 137 (43.9) 157(50.3) aMean±SD. bMedian age. cNumber (%). dReference values for hematological markers: Hb>100 g/L, Zinc Protoporphyrin <80 µmol per mole of heme, serum Transferrin ≤8.3 µg/ml, serum Ferritin >12µg/L, Red Cell Distribution Width ≤14%. eMedian (5th; 95th percentile). Overall, children in the PP group had 6% lower rate of diarrhea (95% CI: −1 to 12%; p = 0.08) compared to children in Co group. Ancillary analysis based on age revealed a significant age interaction, therefore making overall rate reduction less meaningful; for children aged 12 to 24 months rate of diarrhea was [1% lower (95% CI: −11% to 11%), p = 0.91] and children aged >24 months it was [10% lower (95% CI: 2% to 17%), p = 0.02]; p-value for test of difference = 0.03. The incidence of dysentery was 21% lower (95% CI: 0 to 38%; p = 0.05) in the prebiotic and probiotic group than the control group (Table 3). 10.1371/journal.pone.0012164.t003Table 3BODY.EFFECT OF PREBIOTIC OLIGOSACCHARIDE AND PROBIOTIC :Bifidobacterium lactis HN019 fortified milk on common childhood morbidities. PP group (n = 312) Co group (n = 312) Odds Ratio (95% CI) p value Actual numbers Episodes per child year Actual numbers Episodes per child year Gastrointestinal morbidity Diarrhea episodes (1–4 y) 1641a 6.21b 1697a 6.61b 0.94 (0.88–1.01) 0.08  ≤24 mo 603 2.3 563 2.2 0.99 (0.89–1.11) 0.91  >24 mo 1038 3.92 1134 4.41 0.90 (0.83–0.98) 0.02 Dysentery episodes 125 0.47 154 0.6 0.79 (0.62–1.00) 0.05 Respiratory morbidity Pneumonia episodesc 90 0.34 115 0.45 0.76 (0.58–1.00) 0.05 Severe ALRI episodesd 34 0.13 51 0.20 0.65 (0.42–1.00) 0.05 Febrile illness and others Days with severe illness (1–4 y) 473 1.8 550 2.14 0.84 (0.74–0.95) 0.004  ≤24 mo 153 0.58 177 0.69 0.80 (0.65–0.99) 0.05  >24 mo 320 1.21 373 1.5 0.85 (0.73–0.98) 0.03 Days with ear discharge 1550 5.87 1613 6.3 0.93 (0.87–1.00) 0.06 Days with high fever 2798 10.6 2865 11.2 0.95 (0.90–1.00) 0.05 Measles 5 0.02 10 0.04 0.49 (0.17–1.42) 0.19 Doses of antibiotics consumed 7402 28.02 7625 29.7 0.94 (0.91–0.97) <0.001 aActual numbers. bEpisodes per child year. cField based Pneumonia Definition a) If RR was > = 50/min, or b) RR was > = 40/min but was accompanied by lower chest in-drawing or temperature of ≥37.7°C. dRR> = 50/min. There was a 24% reduction (95% CI: 0 to 42%) in incidence of pneumonia (field based definition) and 35% reduction (95% CI: 0 to 58%) in severe ALRI in the prebiotic and probiotic fortified group compared to the control group. Although statistically significant, the confidence interval of this difference was wide and compatible with small or no difference at upper bound of confidence interval (Table 3). The prevalence of severe illness among children consuming prebiotic and prebiotics-fortified milk was 16% (95% CI: 5 to 26%; p = 0.004) lower than the control group. This was similar among children 1 to 2 years [20% lower (95% CI: 1 to 35%); p = 0.05], and 2 to 4 years [15% lower (95% CI: 2 to 27%); p = 0.03] (Table 3). Children in the prebiotic and probiotic fortified group showed a statistically significant 5% fewer days with high fever and 7% lower prevalence of ear discharge compared to control group. The antibiotics usage was [6% (95% CI: 3 to 9%), p<0.001] less among children consuming fortified milk (Table 3). Sub group analyses based on breast-feeding, malnutrition and anemia have been presented in supplementary tables (see Tables S1, S2, S3, S4, S5 and S6). BODY.DISCUSSION: This study, reports the first large randomized controlled trial, evaluating effect of providing combination of prebiotic and probiotic in milk for one year on both gut and non-gut related illnesses among children in a community based setting. We found a significant beneficial effect on dysentery, pneumonia and febrile illnesses. Effect on diarrhea was restricted to children aged >24 months. Although age interaction was statistically robust, given the exploratory nature of this finding, results need to be interpreted with caution. Health effects of probiotics can vary by the specific probiotic used. Bifidobacterium lactis HN019 used in this study has shown extensive safety and immune-stimulant activity in animal models including impact in animal models for E.coli and rotavirus [28]–[30]. Immune-stimulant activity among healthy adult volunteers, with no notable adverse health events has been documented [31]–[33]. Both intervention and control group children consumed similar quantity of milk, and milk in both groups was iso-energic, with identical macronutrient quality and quantity as well as quantity of vitamins and minerals. The only difference was the milk for children allocated to the prebiotic and probiotic group delivered additionally 2.4 g/day of prebiotic oligosaccharide and 1.9×107 c.f.u/day of probiotic Bifidobacterium lactis HN019. The results in this study need to be interpreted as the effects of the combination of oligosaccharide and Bifidobacterium lactis HN019. In addition, in the interpretation of results we need to consider that the base milk used was fortified with iron, zinc and vitamins and improved in nutrient composition (Table 1). Although unlikely but, we cannot exclude the possibility that this is important for success of intervention and therefore effects observed may not be same if prebiotic and probiotic were added to regular unfortified milk. Enrolled children were randomly allocated to the two intervention groups, and the participants, health workers and investigators were masked to group allocation. The similar pattern of compliance between intervention and control groups further supports the belief that masking was very good. As the study relied on active, biweekly follow up by household based surveillance, this would have identified the occurrence of almost all the clinical outcomes of interest, thereby further limiting the possibility of a reporting bias. Although there is substantial evidence for the role of probiotics in diarrhea, majority of that evidence is either from treatment of acute diarrhea [7], [9], [10], [12], persistent diarrhea [13] or antibiotic associated diarrhea [14]–[16] and occurrence of nosocomial infections [34]–[36]. This evidence cannot be extrapolated to prevention of diarrhea in healthy children. The overall reduction in incidence of diarrhea of 6% in this study is similar to, the only other large community based trial, conducted among Peruvian infants and young children [23]. The results of a significant reduction in dysentery episodes, and diarrheal episodes in children 2–4 years is consistent with results from other published randomized controlled trials evaluating prevention of acute diarrhea acquired in day care centers [20], [37]–[41]. The significant differential effect of probiotic on the incidence of diarrhea among younger and older children may explain variations in the results of the previous studies. The lack of effect in children below 24 months of age could be due to one or more of the following: a) difference in the constitution of the gut flora among children >24 months of age, b) a shift in Th1 and Th2 balance. During infancy, the cellular immune system is maturing with a shift from Th2 predomination at birth to Th1 predomination by second year of life. The effect of probiotic may be limited in infants due to intrinsic limitations in the capacity of infants to produce interferon and other Th1 interleukins (IL-2, IL12), c) Breastfeeding among younger children may have modified the effect of probiotic, as studies have suggested greater benefits of prebiotic and probiotic in non-breastfed children as compared to breast fed children [23]. We did not observe significant difference in diarrhea reduction between breast fed and non breast fed children in the age group of 12–24 months; however given lack of power we also cannot exclude it. Although we did not evaluate etiology of diarrhea in this study, previous evaluation have shown rotavirus to be responsible for 2.3% of cases, enterotoxigenic Escherichia coli and enteropathogenic Escherichia coli as major causes with 13.5% and 6.3% of cases, while for dysentery Salmonella species and Shigella species account for 3.2 and 1.8% of cases [42]. Cholera is not endemic in this population. A reduction in incidence and prevalence of febrile illness, pneumonia, severe ALRI, marginal reduction in ear infections and requirement for antibiotics is consistent with findings from the only three studies which have evaluated prevention of similar morbidity syndromes in healthy children [18]–[20]. However, these studies, for many of these outcomes documented trends only, due to lack of statistical power. This study which is the largest reported thus far, implemented an active home based surveillance for morbidity and had a follow up of full one year which potentially provided sensitive estimation of the morbidity. The beneficial effects documented in this study are multi-systemic, indicating that the underlying mechanism for the beneficial effects most likely was due to improved immune response to viral and bacterial infections. Improvement in immunity could have been mediated through improved production of antimicrobial substances, attachment in intestinal mucosal sites, inhibition of the attachment and growth of pathogenic organisms by achieving competitive exclusion and microbial balance leading to regeneration of gut epithelium and consequently resulting in better absorption of nutrients [43]–[46]. However given the variation in effects of probiotics upon such immune mechanisms, the observed effects should be interpreted as effects of the preparation used in this study and not generalized to all prebiotic and probiotic combinations. Reducing the preventable childhood illnesses among preschool children in developing countries is an important public health goal, that would not only impact mortality by breaking malnutrition cycle but would also impact better development of children. The findings of this study suggest that fortification with prebiotic and probiotic together may provide one of the potential interventions to reduce the burden of common childhood morbidities. However, before any public health recommendations are made, these results need to be confirmed in varied settings and with locally available probiotic strains. BODY.SUPPORTING INFORMATION: Table S1Episodes of common childhood morbidities for children who were breast fed. (0.04 MB DOC) Click here for additional data file. Table S2Episodes of common childhood morbidities for children who were non breast fed. (0.04 MB DOC) Click here for additional data file. Table S3Effect of prebiotic oligosaccharide and probiotic Bifidobacterium lactis HN019 and fortified milk on common childhood morbidities (among anemic children). (0.04 MB DOC) Click here for additional data file. Table S4Effect of prebiotic oligosaccharide and probiotic Bifidobacterium lactis HN019 and fortified milk on common childhood morbidities (among non anemic children). (0.04 MB DOC) Click here for additional data file. Table S5Effect of prebiotic oligosaccharide and probiotic Bifidobacterium lactis HN019 and fortified milk on common childhood morbidities (among malnourished children). (0.04 MB DOC) Click here for additional data file. Table S6Effect of prebiotic oligosaccharide and probiotic Bifidobacterium lactis HN019 and fortified milk on common childhood morbidities (among non malnourished children). (0.04 MB DOC) Click here for additional data file. Checklist S1CONSORT Statement 2001 - Checklist. (0.06 MB DOC) Click here for additional data file. Protocol S1Protocol for Efficacy Study of Milk Fortified with Bifidobacillus lactis HNO19 and Oligosaccharides or Zinc and Iron and other Micronutrients. (0.27 MB DOC) Click here for additional data file.

TITLE: Assessing Water Filtration and Safe Storage in Households with Young Children of HIV-Positive Mothers: A Randomized, Controlled Trial in ZambiaWater Filters for Children of HIV-Positive Mothers ABSTRACT.BACKGROUND: Unsafe drinking water presents a particular threat to people living with HIV/AIDS (PLHIV) due to the increased risk of opportunistic infections, diarrhea-associated malabsorption of essential nutrients, and increased exposure to untreated water for children of HIV-positive mothers who use replacement feeding to reduce the risk of HIV transmission. This population may particularly benefit from an intervention to improve water quality in the home. ABSTRACT.METHODS AND FINDINGS: We conducted a 12-month randomized, controlled field trial in Zambia among 120 households with children <2 years (100 with HIV-positive mothers and 20 with HIV-negative mothers to reduce stigma of participation) to assess a high-performance water filter and jerry cans for safe storage. Households were followed up monthly to assess use, drinking water quality (thermotolerant coliforms (TTC), an indicator of fecal contamination) and reported diarrhea (7-day recall) among children <2 years and all members of the household. Because previous attempts to blind the filter have been unsuccessful, we also assessed weight-for-age Z-scores (WAZ) as an objective measure of diarrhea impact. Filter use was high, with 96% (596/620) of household visits meeting the criteria for users. The quality of water stored in intervention households was significantly better than in control households (3 vs. 181 TTC/100 mL, respectively, p<0.001). The intervention was associated with reductions in the longitudinal prevalence of reported diarrhea of 53% among children <2 years (LPR = 0.47, 95% CI: 0.30–0.73, p = 0.001) and 54% among all household members (LPR = 0.46, 95% CI: 0.30–0.70, p<0.001). While reduced WAZ was associated with reported diarrhea (−0.26; 95% CI: −0.37 to −0.14, p<0.001), there was no difference in WAZ between intervention and control groups. ABSTRACT.CONCLUSION: In this population living with HIV/AIDS, a water filter combined with safe storage was used correctly and consistently, was highly effective in improving drinking water quality, and was protective against diarrhea. ABSTRACT.TRIAL REGISTRATION: Clinicaltrials.gov NCT01116908 BODY.INTRODUCTION: Unsafe drinking water is a major cause of diarrheal death and disease, especially for young children in low-income countries and people living with HIV/AIDS (PLHIV). The 33 million PLHIV worldwide - including almost 1 million living in Zambia [1] - are especially vulnerable to diarrheal disease caused by opportunistic infections from waterborne pathogens, such as Cryptosporidium spp. [2], [3]. Diarrheal disease may lead to intestinal malabsorption so that PLHIV on antiretrovirals (ARVs) are not acquiring their essential nutrients and therapeutic dosages of medications [4], [5], [6]. Furthermore, diarrheal disease and unsafe drinking water may be particularly debilitating for children born to HIV-positive mothers. Young children born to HIV-positive mothers are at greater risk of mortality, morbidity, and malnutrition, which may be aggravated by enteric infection [7], [8], [9]. Safe water is critical for HIV-positive mothers who choose to replacement feed in order to prevent transmission of the virus via breast milk; “safe water and sanitation” is the first condition for replacement feeding in the new World Health Organization (WHO) guidelines [10]. Current WHO guidelines for infant feeding for HIV-positive women recommend that virtually all women breastfeed their children for up to 2 years while either the mother or child is on ARVs [10]; the risks of diarrheal disease and malnutrition outweigh the risks of HIV transmission in the majority of low-income settings. Even for mothers who choose to breastfeed, infants may be exposed to waterborne pathogens; exclusive breastfeeding is less common among HIV-positive mothers [11] and water treatment has been found to reduce diarrhea even among breastfed children [12]. Finally, young children who do contract the HIV virus will be more susceptible to water-related pathogens because of a weakened immune system and may particularly benefit from improved environmental conditions. Our previous research in Zambia found that children <2 years born to HIV-positive mothers are particularly at risk of diarrheal disease. In our cross-sectional study, 26% of children <2 years had diarrhea in the past week and bacterial contamination of drinking water was found in 70% of households [13]. Children were more likely to have diarrhea if they had been given water in the past two days, suggesting that diarrheal disease may be at least partially attributable to unsafe drinking water. Additionally, diarrhea in children was significantly associated with mother's diarrhea, which is of particular concern in HIV-affected areas; mothers with HIV may be more likely to have diarrhea [2] and consequently more likely to pass diarrhea onto their children. Therefore, for children born to HIV-positive mothers in low-income settings, water quality interventions may be particularly critical. Improving household drinking water quality through household water treatment and safe storage (HWTS) has been shown to have the potential to significantly reduce diarrheal disease [14], [15], [16]. International organizations including USAID, the World Bank, and WHO have recently called for an integration of water and sanitation activities in HIV/AIDS programs [17], [18], [19], [20], and the number of programs including HWTS for PLHIV is increasing [21], [22], [23], [24], [25], [26]. However, despite these programs, there is relatively little evidence demonstrating the health impact or examining use of HWTS interventions for PLHIV. Only one study has assessed the health impact of HWTS for PLHIV in a low-income setting in the form of a randomized, controlled trial. This trial in Uganda found that PLHIV with a household chlorination technology had 25% fewer diarrhea episodes and 33% fewer days with diarrhea compared to the control group, though diarrhea reductions were not significant for children under five [27]. Other observational studies of household chlorination interventions have found significant associations with diarrhea reductions in Nigeria among adults with HIV/AIDS [28] and in Kenya among infants born to HIV-positive mothers [29]. However, these studies and the majority of HWTS programs for PLHIV have been in the form of chlorination products [30], [31], [32], which do not inactivate or remove the full array of waterborne pathogens (such as Cryptosporidium spp.) unless combined with other treatment mechanisms [33]. Furthermore, there are questions about whether HWTS interventions are used correctly and consistently over an extended period of time [34], [35]; this study is primarily designed to examine HWTS use, which is vital to the success of HWTS programs. We undertook a randomized controlled trial to assess a gravity water filter combined with local jerry cans for safe storage. Specifically, we examined 1) the use of the HWTS, both for children <2 years and all household members, 2) the microbiological performance of the HWTS intervention, measured as thermotolerant (fecal) coliforms (TTC), a well-established WHO indicator organism for fecal contamination [36], and 3) the impact of the intervention on the longitudinal prevalence of diarrhea among children <2 years and all household members, measured both as reported by the primary caretaker and by the weight-for-age z-score (WAZ) of children <2 years —a potential measure for reported diarrhea [37]. BODY.METHODS: The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. BODY.STUDY DESIGN AND SAMPLE SIZE: A randomized, controlled trial was designed to assess use, microbiological performance, and health impact of a household filtration intervention over 1 year. This study followed an open (non-blinded) design because previous attempts to blind the same intervention (LifeStraw Family filter) in the Congo were unsuccessful; the “placebo” provided to control households removed approximately 1 log (90%) of fecal contamination, potentially due to the formation of a biofilm, and the authors concluded that blinding this filter is not likely to be possible [38]. We estimated a sample size of 50 households per arm (100 total) would allow us to estimate use with a precision of at least ±15% with 20% loss to follow-up, assuming at least 70% use [38]. Additionally, 10 HIV-negative mothers and their households were included in each arm (20 total, an additional 20% of households). This figure represents a balance between the need to reduce potential stigma of participation and the cost and inconvenience to additional participants. Because recruitment occurred over an eight month period, the length of possible follow-up depended on the time of enrollment, up to 12 months. With this sample size, we had 80% power to detect a 40% reduction in diarrhea prevalence. BODY.STUDY LOCATION: From our previous work [13], Chongwe District, Zambia was identified for this study based on the lack of piped water supply systems, inadequate water quality, and presence of active health clinics. The project sites included two neighboring compounds, Kasisi and Ngwerere in Chongwe District, both approximately 30 min–1 hour from central Lusaka, Zambia. Neither Kasisi nor Ngwerere were serviced by municipal piped water systems at the time of this study. BODY.PARTICIPANT ELIGIBILITY AND ENROLLMENT: Children <2 years born to eligible HIV-positive mothers were targeted by recruiting and enrolling their mothers. Women were eligible to participate in the trial if they (i) had a child aged 6 to 12 months at the beginning of the trial, (ii) reported that they were HIV-positive (or HIV-negative) confirmed with antenatal clinic records and willing to disclose their status to our study team, and (iii) resided in a household located within the catchment areas of the Ngwerere or Kasisi health clinics in Chongwe district, Zambia and did not plan to move in the next 12 months. Health clinic staff identified potentially eligible women consecutively through under-five clinics and ART programs at their respective health clinics and referred them to our field team. HIV status of the children <2 years was recorded as reported by the mother. BODY.INTERVENTION: Each intervention household received one LifeStraw Family filter and two 5-L safe storage containers. The LifeStraw Family is a novel HWTS filtration technology developed by Vestergaard-Frandsen SA that uses ultrafiltration in the form of a hollow-fiber cartridge to remove pathogens from drinking water [39]. To operate, untreated (influent) water is poured into a 2.5 L container, flows down a 1 m long tube designed to provide head pressure, and through the ulrafiltration cartridge where is it dispensed via tap (effluent). In addition to the filter, we provided two locally-procured 5-L jerry cans (Merco Ltd, Ndola, Zambia) for safely storing water following treatment. Households that were allocated to the intervention group received the filter and training on use and maintenance by our fieldworkers, who were previously trained by the filter manufacturer. Households allocated to the control group were instructed to continue usual practices throughout the study and were allocated filters and storage containers with subsequent training at the end of the study in August 2011. BODY.BASELINE SURVEY AND RANDOMIZATION: At enrollment, baseline data were collected on demographics, sanitation facilities, hygiene practices, water sources and treatment practices, and feeding practices for children <2 years. For each household, baseline water samples were collected from drinking water sources and stored drinking water in the home. Households were randomly allocated using a computer random number generator to either a) the intervention group receiving the LifeStraw Family filter and storage containers, or b) the control group. The randomization was stratified by maternal HIV-status and catchment area (either Ngwerere or Kasisi) in blocks of 8 maximum. The randomization was conducted by the trial manager (RP) who was not involved in the enrollment of participants, and fieldworkers were not involved in the randomization. Participants were recruited from April–December 2010, and followed for 7–12 months depending on time of recruitment. Households were considered to have completed the trial that continued until July 2011, regardless of the time of recruitment; total possible follow-up visits were calculated based on the time from enrollment until July 2011. Households were visited monthly; visits were unannounced and the field team made a repeat visit if the mother was not at home. Although we cannot rule out the potential of courtesy bias assessments of compliance, we took steps to minimize this by making all visits unannounced and sampling water quality, an objective measure. BODY.OUTCOME MEASURES: BODY.USE: Households were followed monthly to obtain information on filter use and acceptability. Households were classified as “reported users” if 1) the filter was observed in household at the time of visit, 2) the storage vessel contained water reported to be treated at the time of visit, and 3) the respondent reported using the filter on the day of or day prior to the day of visit. Households were classified as “confirmed users” if, in addition to these three criteria, there was at least a 1 log10 TTC improvement in their stored household water over their unfiltered water, or stored water quality was <10 TTC/100 mL. “Exclusive use” was defined as not drinking any unfiltered water in the day of or day prior to the interview as reported by the mother. The acceptability of the technology was evaluated through monthly household surveys. BODY.WATER QUALITY: Water samples were collected during monthly visits. For the stored drinking water, the respondent was asked if there was any drinking water in the house and samples were collected from the vessel that the householder identified for drinking. For control households, only stored drinking water was collected. For intervention households, water samples were collected of i) unfiltered water stored in the home (influent water), ii) filtered water immediately after filtration (effluent water), and iii) stored water that the household reported to be filtered, if available. Samples (125-mL) were collected in sterile Whirl-Pak™ Bags (Nasco International, Fort Atikinson, WI, USA) containing a tablet of sodium thiosulfate to neutralize any disinfectant, placed on ice, and processed within 4 hours of collection to assess levels of TTC/100 mL at the University Teaching Hospital, Zambia. Microbiological assessment was performed using a membrane filtration method with membrane lauryl suphate medium using using a DelAgua field incubator (Robens Institute, University of Surrey, Guildford, Surry, UK) in accordance with the Standard Methods [40]. After piloting the assay procedures, we elected to use full 100 ml samples for filtered and filtered & stored samples (intervention households) and 10-fold diluted samples for unfiltered samples (intervention and control households) to minimize the number of samples that yielded plates with colonies that were too numerous to count (TNTC). Where plates were TNTC, we ascribed a value of 500 TTC to such plates; this is a conservative estimate of the upper detection limit considering up to 1500 TTC were counted per plate. Baseline samples were also tested for free and total chlorine residuals using a Hach color-wheel test kit (Hach Company, Loveland, CO, USA). BODY.DIARRHEA LONGITUDINAL PREVALENCE: At all monthly visits, the mother was asked whether each household member experienced any diarrhea in the past 7 days. Diarrhea was measured as longitudinal prevalence (the proportion of weeks with diarrhea divided by the number of weeks under observation) [41]. Diarrhea was defined according to the WHO definition of 3 or more loose stools within a 24-hour period [42], [43]. Mothers who reported diarrhea were also asked whether the episode extended for 14 days or longer in order to assess persistent diarrhea. BODY.WEIGHT-FOR-AGE Z-SCORES (WAZ): Children <2 years were weighed during monthly visits on baby scales (Seca Model 384, Chasmors, London, UK) according to standard protocol [44]. During weight measurements, children were only wearing a minimum of light clothing without shoes. Children were weighed a minimum of twice during every visit to verify the weight measurement; if the two measurements were not equal (particularly from child movement), the child was weighed a third time and the confirmed weight was recorded. Date of birth was verified on the child's health card to calculate WAZ. BODY.DATA MANAGEMENT AND ANALYSIS: Data were double-entered into EpiData 3.1 and analyzed using Stata 12. The analysis plan was finalized before the data were examined. WAZ scores were calculated using the WHO growth reference data. Socioeconomic status was measured using an asset index created by combining data on household possessions and characteristics based on asset questionnaires used in the Zambia Demographic and Health Survey [45]. Data were analyzed on an intention-to-treat basis in order to estimate the effectiveness of supplying households with the intervention, regardless of filter use. The data from households with HIV-negative mothers were included in all analyses unless stated otherwise. To assess acceptability and filter use, we tabulated data for all visits combined, and separately for the ‘final’ visit, defined as the final follow-up visit for households that completed the trial. To assess the effect of the intervention on water quality, TTC counts during follow-up were compared using random effects linear regression to account for repeated observations within households. TTC counts were normalized with log10 transformations; a value of 1 was added to all TTC levels before transformation to account for samples with TTC values of zero, log10(TTC level+1). Microbiological filter performance was calculated as the difference of the log of the influent concentration and log of the effluent concentration. All water quality analyses assumed that intervention households were drinking unfiltered water if stored filtered water was not available at the time of visit. The effect of the intervention on diarrhea longitudinal prevalence was examined using binomial regression with a log link function and robust standard errors, with generalized estimating equations (GEE) to account for correlation of repeated measures within individuals [46]. In the analysis of diarrhea for all household members, we accounted for clustering at the household level, since this adequately accounted for within-individual correlation. The effect of the intervention on WAZ was assessed using random effects linear regression to account for repeated observations within individuals. In a secondary analysis we controlled for WAZ at baseline. To examine the relationship between WAZ and diarrhea, we used random effects linear regression to account for repeated measures and adjusted for baseline WAZ. To assess the relationship between water quality and diarrhea longitudinal prevalence, we used binomial regression with a log link function and robust standard errors with GEE to account for correlation of repeated measures. Water quality results were transformed to log10(TTC level+1), to account for samples with TTC values of zero. Adjusted analyses controlled for age and trial arm, since both were strongly associated with diarrhea. Predicted probabilities of diarrhea from the unadjusted and adjusted models were calculated at fixed values of log10 TTC and plotted. We used fractional polynomials to examine the shape of the relationship of water quality (log10 TTC) with log diarrhea prevalence, using a set of defined powers (−2, −1, −0.5, 0.5, 1, 2 and ln(x)) and a maximum of two power terms in the model. Models were adjusted for intervention arm. The differences in model deviances were compared; the linear model was used if the improvement in fit was not statistically significant at p<0.05. The relationship between water quality and WAZ was assessed with random effects linear regression accounting for repeated measures and adjusted for baseline WAZ; adjusting for baseline WAZ accounts for genetic variability and events prior to the intervention. To examine the effect of the intervention on mortality, we used a Cox Proportional hazard model to estimate mortality rates. BODY.ETHICS: This study was approved by the Biomedical Research Ethics Committee of the University of Zambia and the London School of Hygiene and Tropical Medicine Ethics Committee, and registered with clinicaltrials.gov (NCT01116908). Participants were provided with verbal and printed details of the study in the local language; informed, written consent was obtained from all participating mothers for their respective households. Measures were taken to ensure confidentiality for all participants. If members of participating households were found to be in need of health care during the household visits, they were referred to health clinics. At the conclusion of the study, the results were disseminated to all participants in community meetings, and all control households received the intervention. Besides the intervention, households were not given incentives to participate. BODY.RESULTS: BODY.STUDY POPULATION: 141 mothers were screened; 17 (12%) were ineligible and 4 (3%) refused to participate (Figure 1p = 0.47). There were 3/61 (5%) deaths in children <2 years in the intervention arm and 6/60 (10%) in the control (p = 0.28). 10.1371/journal.pone.0046548.g001Figure 1BODY.RECRUITMENT FLOW DIAGRAM.: Among children <2 years, data were collected for 82% (1138/1382) of possible person-weeks of diarrhea. Baseline characteristics were distributed evenly between the trial arms, with the exception of mother's marital status, sex of child <2 years, and reported diarrhea (Table 1). Only 12% (14/121) of children <2 years were reported to be HIV-positive, 50% (61/121) were negative, and 38% (46/121) had not been tested by the end of our study. 10.1371/journal.pone.0046548.t001Table 1BODY.SELECTED BASELINE CHARACTERISTICS OF INTERVENTION AND CONTROL HOUSEHOLDS.: Intervention Control Demographics Number of households 61 (51%) 59 (49%) Number of households in Ngwerere 27 (44%) 27 (46%) Number of households in Kasisi 34 (56%) 32 (54%) Number of people 299 300 Number of children 6–12 months 61 60 Median persons per household (range) 5 (2–10) 5 (2–10) Median mother's age (range) 28 (17–44) 30 (18–41) Mother is married or living with partner 54 (89%) 41 (69%) Mother has some education 49 (80%) 48 (81%) Mother is HIV-positive 51 (84%) 49 (83%) Mother on antiretroviral therapy 17 (28%) 16 (27%) Socioeconomic quintiles Lowest 3 (5%) 14 (24%) Low 4 (7%) 4 (7%) Middle 20 (33%) 13 (22%) High 21 (34%) 16 (27%) Highest 13 (21%) 12 (20%) Water Source Piped into home or yard 3 (5%) 3 (5%) Public standpipe 12 (20%) 10 (17%) Borehole 7 (11%) 11 (19%) Protected dug well 3 (5%) 7 (12%) Unprotected dug well 35 (57%) 27 (46%) Surface Water 1 (2%) 1 (2%) Water, Sanitation & Hygiene practices Report usually treating water 12 (20%) 13 (22%) Report usually chlorinating 12 (20%) 11 (19%) Report usually boiling 0 (0%) 3 (5%) Had treated water at time of visit 6 (10%) 7 (12%) Water storage container covered 54 (89%) 49 (83%) Use cup used to draw water from storage container 10 (16%) 17 (29%) Improved sanitation facility 15 (25%) 16 (28%) Soap present in household 27 (44%) 32 (54%) Water Quality Household TTC Geometric Mean (95%CI)1 272 (157–470) 317 (179–564) Source TTC: Geometric mean (95% CI)1 117 (72–190) 193 (114–328) Household free chlorine ≥0.2 mg/L1 1 (2%) 0 (0%) Source free chlorine ≥0.2 mg/L1 0 (0%) 0 (0%) Diarrhea in all household members Diarrhea in the past 7 days1 44 (15%) 27 (9%) Persistent diarrhea in the past 7 days1 10 (3%) 5 (2%) Persistent diarrhea in the past 7 days1 10 (3%) 5 (2%) Children <2 years Median age (SD) in months at recruitment 7.5 (1.9) 6.9 (1.9) Male 34 (56%) 22 (37%) Diarrhea in past 7 days 18 (30%) 17 (28%) Persistent diarrhea in past 7 days 4 (7%) 4 (7%) HIV-positive, if known 2 (3%) 1 (2%) Mean (SD) Weight-for-age z-score −0.81 (1.42) −0.97 (1.59) Currently breastfed 49 (80%) 46 (77%) Study Follow up Time Mean follow up (min-max) in months 11.2 (7–12) 11.3 (8–12) 1Data are missing for 1 household on stored water TTC, 2 households on source water TTC, and 3 households on stored water chlorine residual. Three individuals are missing data on reported diarrhea and 5 individuals missing data on persistent diarrhea. BODY.FILTER USE: Most households used the filters throughout the study (Table 2). Households were classified as reported users in 96% (596/620) of all household visits and as confirmed users in 87% (540/620) visits. If we were to restrict our definition of confirmed user to only those that had at least 1 log10 removal of TTC, 82% (507/620) of intervention households would still be considered confirmed users. Among households that did not meet the criteria of confirmed users, 4% (24/620) visits had stored water of somewhat better water quality compared to unfiltered water (<1 log10) and therefore may have been actually using the filter. In instances when households did not have stored filtered water at the time of visit (3% of all visits, 16/622) the mother reported that she did not have time to filter the water. Only 3/61 (<5%) of filters had to be replaced during the study; 1 clogged and 2 were eaten by rats along the filter tubing 10.1371/journal.pone.0046548.t002Table 2BODY.FILTER USE AND ACCEPTABILITY AMONG INTERVENTION HOUSEHOLDS.: Final Visit All Visits N = 53 % N = 627 % Filter Use Reported user1 51/53 96% 596/620 96% Confirmed user2 49/53 92% 540/620 87% Exclusive use by mother today/yesterday3 49/53 92% 591/624 95% Exclusive use by child <2 years today/yesterday3 48/50 96% 171/184 93% Filter present in household 53/53 100% 625/626 >99% Filtered water for drinking today or yesterday 53/53 100% 606/624 97% Currently have filtered water stored4 51/53 96% 606/622 97% Always used filter in past week 53/53 100% 620/623 >99% Stored filtered ≥1 log10 TTC lower than unfiltered water, or <10 TTC/100 mL 49/51 96% 557/604 92% Median volume of filtered water used per day (range)5 5 L (5 L) 5 L (2.5–20 L) Mother is responsible for filter 53/53 100% 617/626 99% What people like best about the filter Provides safe water 40/53 75% 337/618 55% Improves water taste 7/53 13% 129/618 21% Provides good water 5/53 9% 143/618 23% Easy to Use 1/53 2% 11/618 2% What people like least about the filter Nothing – everything is ok 53/53 100% 615/621 99% Flow rate is too slow 0/53 0% 3/621 <1% Filter is broken/has a problem 0/53 0% 2/621 <1% Doesn't provide enough water 0/53 0% 1/621 <1% Filter Maintenance 6 Backwashed today or yesterday 52/53 98% 601/624 96% Cleaned pre-filter today or yesterday 52/53 98% 603/624 97% Water Storage Using storage container provided 53/53 100% 623/625 >99% Storage container capped 52/53 98% 623/624 >99% Only store filtered water in supplied containers 51/53 96% 610/624 98% 1Households were classified as “reported users” if 1) the filter was observed at the time of visit, 2) the storage vessel contained water reported to be treated, and 3) the respondent reported using the filter today or yesterday. 2Households were classified as “confirmed users” if in addition to the criteria for reported users, there was at least a 1 log10 TTC improvement in stored household water over unfiltered water, or stored water quality was <10 TTC/100 ml. 3Exclusive use was defined as not drinking any unfiltered water today or yesterday. For all households that did not report exclusive use, the reason for drinking unfiltered water was that they were away from home. For children <2 years, 3 children in intervention arm died so there are data missing at the final visit. Exclusive use for children <2 years data were only collected in the last quarter of the study period. 4Mothers that didn't have filtered water reported that they did not have time to filter. 55 L is 1 container provided; all households reported 1 container (2 households missing data). 6Households were instructed to backwash and clean the pre-filters daily, as recommended by the manufacturer. Mothers reported exclusively using the filters in 95% (591/624) of all visits. For children <2 years, exclusive use was reported in 93% (171/184) of all visits. Reasons for not using the filter exclusively were that the mother or children were away from home, such as visiting relatives or at church. Almost all households (>99%, 623/625 visits) reported using the storage containers provided to store filtered water. Results at the final visit were similar to those at all visits (Table 2). BODY.WATER QUALITY: Unfiltered water had a geometric mean of 190 TTC/100 mL (95% CI: 147–245 TTC/100 mL), with 60.3% (720/1194) of samples over 100 TTC/100 mL (Figure 2). 3.3% of unfiltered intervention group water samples and 4.5% of unfiltered control group water samples yielded plates that were TNTC; no filtered samples and filtered and stored samples resulted in TNTC plates. Unfiltered water did not differ significantly between the intervention and control groups (geometric mean 199 vs. 181 TTC/100 mL, respectively, p = 0.61). In intervention households, water quality was significantly better in filtered water (geometric mean of 1.2 TTC/100 mL; 95% CI: 1.1–1.2 TTC/100 mL) and stored filtered water (geometric mean of 2.7 TTC/100 mL; 95% CI: 2.3–3.0 TTC/100 mL) compared with unfiltered water (Figure 2). The quality of stored drinking water was significantly better in intervention households than control households (geometric mean 3 vs. 181 TTC/100 mL, respectively, p<0.001). In intervention households, the geometric mean removal from influent (unfiltered) to effluent was 2.2 log10 TTC/100 mL (95% CI: 2.1–2.3 log10 TTC/100 mL), corresponding to a 99.4% (95% CI: 99.3–99.5%) reduction. 10.1371/journal.pone.0046548.g002Figure 2BODY.WATER QUALITY TESTING RESULTS.: Unfiltered water is for all households; filtered and stored filtered is only for the intervention arm. BODY.REPORTED DIARRHEA: Diarrhea longitudinal prevalence in children <2 years was 13.6% (72/530) in the control arm and 6.6% (40/608) in the intervention arm, representing a 53% reduction (longitudinal prevalence ratio, LPR = 0.47, 95% CI: 0.30–0.73, p = 0.001) (Table 3 and Figure 3). When restricted to children of HIV-positive mothers, the intervention was associated with a 50% reduction in diarrhea (LPR = 0.50, 95% CI: 0.31–0.80, p = 0.004). For all household members, diarrhea longitudinal prevalence was 3.5% (101/2906) in the control group and 1.6% (50/3168) in the intervention (LPR = 0.46, 95% CI: 0.30–0.70, p<0.001). 10.1371/journal.pone.0046548.g003Figure 3BODY.LONGITUDINAL PREVALENCE OF DIARRHEA IN CHILDREN <2 YEARS AND ALL HOUSEHOLD MEMBERS.: Data for July 2010 are grouped with August 2010, due to follow-up visits commencing the final week of July. 10.1371/journal.pone.0046548.t003Table 3BODY.LONGITUDINAL PREVALENCE OF DIARRHEA IN INTERVENTION AND CONTROL GROUPS.: % Weeks with diarrhea of total possible person-weeks of diarrhea LPR1 (95% CI) P Intervention Control Diarrhea <2 years 6.6% (40/608) 13.6% (72/530) 0.47 (0.30–0.73) 0.001 <2 years, HIV-exposed2 7.1% (36/509) 13.8% (58/419) 0.50 (0.31–0.80) 0.004 <5 years 4.3% (42/967) 8.9% (79/891) 0.51 (0.32–0.80) 0.003 All household 1.6% (50/3168) 3.5% (101/2906) 0.46 (0.30–0.70) <0.001 Persistent diarrhea (≥14 d) <2 years 2.1% (13/608) 0.63 (0.28–1.40) 0.26 <2 years, HIV-exposed2 2.2% (11/509) 3.3% (14/419) 0.61 (0.25–1.49) 0.28 <5 years 1.5% (14/967) 1.9% (17/890) 0.77 (0.35–1.70) 0.51 All household 0.6% (18/3168) 0.7% (21/2904) 0.75 (0.37–1.53) 0.43 LPR = Longitudinal Prevalence Ratio. 1Accounting for repeated measures (children <2 years) and clustering within household (all household data). 2Child is considered HIV-exposed if their mother is HIV-positive. Diarrhea was classified as persistent (≥14 days) in 26.2% (39/149) of reported weeks with diarrhea for all household members and 27.0% (30/111) of reported weeks with diarrhea for children <2 years (Table 3). Most persistent diarrhea occurred in children <2 years (76.9%, 30/39), and the 5 people who had more than one visit with persistent diarrhea were all children <2 years. The intervention resulted in reductions in persistent diarrhea for children <2 years (LPR = 0.63, 95% CI: 0.28–1.40, p = 0.26) and all household members (LPR = 0.75, 95% CI: 0.37–1.53, p = 0.43) though results were not statistically significant. BODY.WEIGHT-FOR-AGE Z-SCORES (WAZ) IN CHILDREN <2 YEARS: There was no evidence of a difference between the intervention and control groups in mean WAZ scores (−1.21 vs. −1.24, respectively, p = 0.92). Adjusting for baseline WAZ did not change this conclusion (−1.18 vs. −1.31, respectively, p = 0.85). Children with concurrent diarrhea had lower average WAZ scores compared to children without diarrhea (−1.46 vs. −1.20, respectively, p<0.001). After adjusting for WAZ at baseline, mean WAZ scores among children <2 years with diarrhea were 0.26 lower than in children without diarrhea (95% CI: −0.37 to −0.14, p<0.001). BODY.WATER QUALITY, DIARRHEA, AND WAZ: There was a suggestion of a positive trend between diarrhea prevalence and household fecal water contamination (Figure 4). The results of the fractional polynomial models showed that the linear model adequately described the relationship between log diarrhea prevalence and log10 TTC. This relationship was significant for all household members (age-adjusted LPR for the increase in prevalence with log10 TTC = 1.29, 95% CI: 1.14–1.45, p<0.001), and for children <2 years (age-adjusted LPR for log10 TTC = 1.20, 95% CI: 1.05–1.39, p = 0.01). Though adjusting for trial arm attenuated the association between water quality and diarrhea, there was still weak evidence of an effect (age- and arm-adjusted LPR for log10 TTC = 1.15, 95% CI: 0.99–1.33, p = 0.07 for all household members; age- and arm-adjusted LPR for log10 TTC = 1.09, 95% CI: 0.92–1.28, p = 0.33 for children <2 years). In contrast, there was no evidence of an association of water quality and WAZ (mean change in WAZ for log10 TTC = 0.00, 95% CI: −0.05 to 0.04, p = 0.93); adjusting for trial arm did not change this conclusion. 10.1371/journal.pone.0046548.g004Figure 4BODY.WATER QUALITY AND DIARRHEA IN CHILDREN <2 YEARS.: Water quality is of stored drinking water (stored filtered water for intervention households and unfiltered water for control households). If intervention households did not have stored filtered water available, it was assumed they were drinking unfiltered water. Both analyses are adjusted for age; adjusting for trial arm is examined separately due to the partial collinearity between trial arm and water quality. Predicted probabilities of diarrhea are from unadjusted and adjusted binomial regression models with log link functions and robust standard errors with GEE to account for repeated measures. Error bars represent 95% confidence intervals. Unadjusted model coefficients: ln(diarrhea prevalence) = −1.25+0.186(log10TTC)+−0.0991(child's age). Adjusted model coefficients: ln(diarrhea prevalence) = −0.868+0.0825(log10 water quality)+−0.0990(child's age)+−0.506 (trial arm). BODY.MORTALITY OF CHILDREN <2 YEARS: During the study, there were 9 deaths, all in children <2 years; 3/61 (5%) in the intervention arm and 6/60 (10%) in the control. The cause of death was recorded as reported by the primary caregiver. In the intervention arm, only one death was gastrointestinal (reported as diarrhea/vomiting); other deaths were from respiratory illness and consuming rat poison. In the control arm, deaths were potentially all gastrointestinal-related (diarrhea/vomiting, diarrhea/malnutrition [3 children], diarrhea/coughing, and malnutrition). All but one were children born to HIV-positive mothers, and two children were known to be HIV-positive. There was no evidence of an impact of the intervention on all-cause mortality among children <2 years (RR = 0.56; 95% CI: 0.13–2.37, p = 0.43), though the study was not designed to detect a difference in mortality as an outcome. BODY.DISCUSSION: To our knowledge, this study is the first randomized controlled trial to examine a HWTS intervention among HIV-positive mothers with young children. Our findings suggest that the intervention was used correctly and consistently, was highly effective in improving drinking water quality, and was protective against diarrhea. Filter use was particularly high in our study; households were using the filters in 96% of visits and use was further confirmed with water quality testing in 87% of visits. Some of the households that did not meet the water quality testing criterion for confirmed use may have been actually using the filter, but recontamination during storage prevented the criterion from being met. It is possible that repeated surveying contributed to increased use of the intervention [47]; some studies have lower uptake of HWTS when delivered programmatically rather than in research-driven efficacy trials such as this [48], [49]. However, there is some evidence that use is particularly high for filtration compared to other HWTS technologies [35], [50]. Previous studies of LifeStraw filters reported 68% use 8 months after distribution (Boisson et al 2010) and 83% use 2 months after distribution LifeStraw Family and LifeStraw personal filters combined) [51]. Furthermore, it is possible that use may be particularly high among HIV-positive mothers with young children because of increased concern and awareness of health; chlorination use has been found to be high among similar populations [25], [29]. A previous field trial of the LifeStraw Family filter in the Congo also reported high rates of use (76%) [38]. However, nearly all householders in that study (83% of adults and 95% of children <5 years) reported also drinking from other untreated sources, compared with only 5% of mothers and 7% of children <2 years in our trial. The large difference in exclusive use may be attributable to the fact that in the Congo trial households were advised to only use water directly from the filter and were not provided with safe storage containers, implying that safe storage containers may be essential to ensure exclusive use of HWTS. At the same time, there is little evidence that the practice of storing water after it is filtered adversely impacted drinking water quality in the home. Diarrhea reductions in our study exceeded the 35–44% commonly found by HWTS [14], [15], [16]. Diarrhea reductions may have been particularly high among our population because of the increased risk of water-related pathogens in households with PLHIV [13], [52], [53] and the performance of the intervention in removing the full array of microbial pathogens. Furthermore, use and exclusive use was high among our population, and there is an increased health impact among high-frequency HWTS users [28], [54]. However, the intervention did not result in significant reductions in persistent diarrhea among children <2 years or all household members. Previous research has found that household water treatment may be more effective in reducing shorter episodes of diarrhea compared to persistent diarrhea [55]. Water quality showed a positive trend with reported diarrhea, both for children <2 years and all household members. Interventions that improve water quality are known to reduce diarrheal disease [15], [56], though the relationship between drinking water quality bacterial indicators and general diarrheal disease is not well established [56], [57], [58]. An observational study in Tanzania found a relationship between health and fecal contamination on hands but not in stored drinking water [59], though a previous trial of a household ceramic filter in Colombia found a significant relationship between water quality and diarrhea [60]. In our study, the suggestion of positive trend between diarrhea and water quality supports our finding that the water quality intervention resulted in a reduction in diarrheal disease; presumably participants would be unable to base reported diarrhea on actual TTC levels in their water considering they were not aware of exact TTC levels. Though we did not find an impact of the intervention on WAZ, we did detect a significant association between WAZ and reported diarrhea. The lack of difference in WAZ between our trial arms despite the reduction in reported diarrhea and the association between WAZ and reported diarrhea merits further discussion. It is possible that reported diarrhea data may be of questionable reliability; open trial designs of self-reported outcomes are subject to bias [61]. We cannot entirely rule out or assess the effects of biased self-reporting of diarrhea. However, the relationship between diarrhea and water quality is well-established and is the basis for international drinking water quality standards [62]. The fact that we observed this same relationship here suggests that our results are not solely attributable to bias self-report. Moreover, we found no association between WAZ and water quality; given that the intervention may only influence WAZ via water quality, the intervention may not be appropriate to improve WAZ. Furthermore, diarrhea and WAZ may be associated primarily due to persistent diarrhea. We did not find a significant reduction in persistent diarrhea in children <2 years (p = 0.26) and a previous trial in Guatemala found that a HWTS intervention mostly prevented short episodes [55], [63]. Therefore, the diarrhea experienced by our intervention arm may have been more persistent compared to the intervention group. This is supported by a stronger relationship between diarrhea and WAZ in the intervention arm than in the control arm (p = 0.003 for interaction); persistent diarrhea is known to impair growth [42], [64]. Though we cannot entirely discount the possibility of reporting bias, WAZ may not be an appropriate measure for diarrhea in HWTS trials, though further investigation is needed. There are some limitations to our study. First, the reliance on self-reported data for diarrhea disease in a non-blinded HWTS intervention trial has previously been criticized [34], [35]. However, the suggestion of positive trend between water quality and diarrhea suggests that most of the self-reported diarrhea may be verifiable. Second, baseline diarrhea prevalence was not evenly distributed between our trial arms for all household members, though this would only result in a conservative estimate of the intervention effect and baseline diarrhea may not be predictive of diarrhea during the intervention period [63]. Third, because we recruited from health clinics, we were not capturing the most vulnerable population that does not have access to health facilities or is too sick to access these services. Finally, our study was conducted in Chongwe District, Zambia and may not be generalizable to other locations with different water quality and practices. Despite these limitations, our findings indicate that HWTS may be particularly beneficial among HIV-positive mothers with young children. Though our study was not designed to examine mortality of children <2 years, our study results and previous research [65] suggest that HWTS may have the potential to reduce mortality in young children. The effect of HWTS on mortality of young children needs to be further explored in the form of a full randomized, controlled trial. 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: Is TIMP-1 immunoreactivity alone or in combination with other markers a predictor of benefit from anthracyclines in the BR9601 adjuvant breast cancer chemotherapy trial? ABSTRACT.INTRODUCTION: Predictive cancer biomarkers to guide the right treatment to the right patient at the right time are strongly needed. The purpose of the present study was to validate prior results that tissue inhibitor of metalloproteinase 1 (TIMP-1) alone or in combination with either HER2 or TOP2A copy number can be used to predict benefit from epirubicin (E) containing chemotherapy compared with cyclophosphamide, methotrexate and fluorouracil (CMF) treatment. ABSTRACT.METHODS: For the purpose of this study, formalin fixed paraffin embedded tumor tissue from women recruited into the BR9601 clinical trial, which randomized patients to E-CMF versus CMF, were analyzed for TIMP-1 immunoreactivity. Using previously collected data for HER2 amplification and TOP2A gene aberrations, we defined patients as "anthracycline non-responsive", that is, 2T (TIMP-1 immunoreactive and TOP2A normal) and HT (TIMP-1 immunoreactive and HER2 negative) and anthracycline responsive (all other cases). ABSTRACT.RESULTS: In total, 288 tumors were available for TIMP-1 analysis with (183/274) 66.8%, and (181/274) 66.0% being classed as 2T and HT responsive, respectively. TIMP-1 was neither associated with patient prognosis (relapse free survival or overall survival) nor with a differential effect of E-CMF and CMF. Also, TIMP-1 did not add to the predictive value of HER2, TOP2A gene aberrations, or to Ki67 immunoreactivity. ABSTRACT.CONCLUSION: This study could not confirm the predictive value of TIMP-1 immunoreactivity in patients randomized to receive E-CMF versus CMF as adjuvant treatment for primary breast cancer. BODY.INTRODUCTION: A number of clinical studies have clearly indicated the superiority of anthracycline-containing chemotherapy over the combination of cyclophosphamide, methotrexate and 5-flourouracil (CMF) in adjuvant treatment of breast cancer [1-3]. However, a significant number of anthracycline treated patients will experience disease recurrence, suggesting that their breast cancers contained tumor cells refractory to adjuvant anthracyclines. Moreover, patients receiving an anthracycline may experience significant toxicity during treatment [4]. With a validated predictive biomarker for anthracycline sensitivity/resistance, it would be possible to direct the toxic adjuvant anthracycline treatment to those patients with the highest likelihood of a treatment benefit while those patients with anthracycline resistant tumors could receive an alternative treatment, for example, a taxane. A number of studies have suggested that breast cancer patients with HER2 positive tumors, those amplified and/or overexpressing HER2, are those obtaining the greatest benefit from the addition of an anthracycline [5]. Similar data have been presented for the Topoisomerase IIα (TIIα) gene copy number (TOP2A) or enzyme, the latter being a target of the anthracyclines [6]. More recently, we have shown that tumor levels of other members of the HER family may be associated with benefit from adjuvant chemotherapy [7]. However, these effects are not substantiated in a recent meta-analysis of multiple trials with data available for HER2 and TOP2A [8]. Emerging data may suggest that novel markers associated with centromeric enumeration probe for chromosome 17 (CEP17) duplication may identify, in part, those patients with anthracycline responsive cancer [9]. However, increasingly, there is recognition of the complex nature of tumor resistance to chemotherapy and the need for multiple markers to stratify patients according to their likelihood of response to chemotherapy. Tissue inhibitor of metalloproteinase 1 (TIMP-1) protein as determined by immunhistochemistry is another potential molecular marker for anthracycline benefit [10]. Preclinical data linked TIMP-1 to protection against chemotherapy-induced inhibition of apoptosis [11,12], and when applying TIMP-1 immunohistochemistry (IHC) to tissue microarrays (TMAs) from the Danish Breast Cancer Cooperative Group (DBCG) 89D prospective randomized adjuvant trial (cyclophosphamide, epirubicin, 5-fluorouracil (CEF) vs CMF), [10], it was demonstrated that women with breast tumors displaying cancer cell TIMP-1 immunoreactivity had similar benefit from adjuvant chemotherapy regardless of the addition of an anthracycline, while women lacking TIMP-1 immunoreactivity in the cancer cells had a significant improved benefit (increased disease free survival (DFS) and overall survival (OS)) when receiving combination therapy with an anthracycline as compared with women who received treatment with CMF [10]. A subsequent study, including the same DBCG 89D patient cohorts, showed that TIMP-1 immunhistochemistry could be combined with the HER2 or TOP2A gene copy number forming a biomarker panel which could predict anthracycline benefit in almost double the number of patients as each of these markers could do separately [13]. In a more recent study [14], including patient samples (TMAs) from the Canadian MA5 study in which patients were randomized to receive either CEF or CMF [14], we reported on a substantial reduction in mortality by CEF compared to CMF in patients with a HER2/TIMP-1 or TOP2A/TIMP-1 responsive profile; however, we could not show a similarly significant reduction in recurrence-free survival events, where a benefit of CEF over CMF was found irrespective of TIMP-1 status. To further test the hypothesis that TIMP-1 in combination with either HER2 or TOP2A copy number can be used to predict benefit from adjuvant chemotherapy, including an anthracycline, we assessed TIMP-1 immunoreactivity in TMAs obtained from the BR9601 study in which patients were randomized to receive either epirubicin followed by CMF (E-CMF) or CMF alone [2]. On all samples, data on tumor genetic alterations of HER2 and TOP2A and protein expression of HER2 and Ki67 were available [7]. BODY.MATERIALS AND METHODS: BODY.PATIENTS: The BR9601 study recruited 374 pre- and post-menopausal women with completely excised, histologically confirmed early breast cancer that had a clear indication for adjuvant chemotherapy according to current UK practice, which relies on the Nottingham Prognostic Index for risk stratification. For further details, please see [2]. The patients were randomized between the standard arm of eight cycles of CMF (intravenous (i.v.) cyclophosphamide 750 mg/m2, methotrexate 50 mg/m2 and 5-fluorouracil 600 mg/m2) every 21 days, and E-CMF (four cycles of 100 mg/m2 of epirubicin every 21 days followed by four cycles of the same CMF regimen). The protocol was approved by central and local ethics committees, and each patient provided written informed consent prior to randomization. The primary outcomes of the BR9601 study were relapse free (RFS) and OS (2). Patients were followed for a mean of 5.30 years (range 2.76 to 8.51 years). For the current analysis, following approval by the central ethics committee (South East Multi-centre Research Ethics Committee), tissue blocks were retrieved from 321 cases (85.8%) among which 288 cases (77.0%) were applicable for the present study. A total of 30 samples were lost on the TMAs and 3 cases had incomplete follow-up. Triplicate tissue microarrays (TMAs) with 0.6 mm2 cores were constructed using cores taken from the middle of the invasive tumor following review by a pathologist (JST). Duplicate TMAs were used for the purpose of this study. BODY.TRIPLE COLOR FLUORESCENT :in situ hybridization (FISH) FISH was performed using a triple-color probe for HER2, Topoisomerase IIα (TOP2A) and chromosome 17 (CEP17) (Abbot Molecular, Maidenhead, Berkshire, UK) as previously described [15-17]. Amplifications were defined as gene:chromosome ratios > 1.5 for TOP2A and > 2.0 for HER2. TOP2A deletions were defined as gene:chromosome ratios < 0.8 [17]. BODY.IMMUNOHISTOCHEMISTRY: The TIMP-1 immunohistochemistry procedure used has previously been described in details [18]. In brief, TMAs were deparaffinized in xylene and rehydrated in graded concentrations of ethanol. For antigen retrieval, the sections were microwave treated in citrate buffer pH6 and endogenous peroxidase activity was blocked by hydrogen peroxide. IHC staining for TIMP-1 was performed overnight at 4°C and used the mouse monoclonal antibody, clone VT7 [19] raised against recombinant human TIMP-1 (0.25 μg/ml). TIMP-1 staining was scored by two experienced observers (AB and EB) blinded to patient identity and outcome. If any TIMP-1 positive cells were evident in the section it was scored as positive, thus following the same procedure as in our previous studies [10,13]. The IHC procedure for Ki67 (clone MIB-1, Dako, Glostrup, Denmark) has been described previously [20]. Staining was scored by a single experienced scorer (AM) blinded to patient identity and outcome and counting the percentage of Ki67 positive cells. For Ki67 13% positivity was used as a cut point for dividing samples into Ki67 low and high, respectively [20], consistent with our previous studies. BODY.STATISTICS: The IBM SPSS (v14) statistical software (IBM corporation, Portsmouth, Hampshire, UK) was used for statistical analysis. Kaplan-Meier estimates of survival were used for analysis of RFS and OS. The Cox's proportional hazard model was used to obtain hazard ratios for relapse or death. When comparing outcomes between the treatment arms within the two groups of patients identified by biomarker expression, formal P-values were not calculated as in most cases, one group was much smaller than the other. The Cox model was instead used to identify statistically significant interactions between biomarkers (expression or gene alterations) and outcome on the different treatments (treatment by marker effect), in models that also included biomarker status (marker effect) and treatment, as covariates. RFS was calculated from the date of randomization to the date of relapse or the date last seen. OS was calculated from the date of randomization to the date of breast cancer specific death or the date last seen. BODY.RESULTS: There were no significant differences in patient baseline characteristics between the overall BR9601 trial population (n = 374) and the population (n = 288) included in this biomarker study (Table 1). Of 288 patients included in the current study, 96 (33.3%) relapsed and 78 (27.1%) died during the follow-up period. A survival analysis of these 288 patients confirmed the advantage of E-CMF over CMF observed in the main trial [2] with HR: 0.57; 95% CI 0.37 to 0.86; P = 0.006 and HR: 0.64; 95% CI 0.41 to 1.01; P = 0.05 for RFS and OS, respectively. All subsequent analyses are restricted to these 288 cases, or sub-sets thereof. Table 1Patient/tumor characteristics from the BR9601 trial and samples retrieved for the current study BR9601 TIMP-1 Analysis Number 374 288 Age 50.9 (44.7 to 56.6) 50.5 (45.0 to 57.0) E-CMF (1) 183 (48.9%) 140 (48.6%) CMF (2) 191 (51.1%) 148 (51.4%) Tumor size (median) 23 (17 to 30) 25 (17 to 30) Positive nodes 2 (1 to 4) 3 (1 to 4) Grade 3 (2 to 3) 3 (2 to 3) NPI 5.30 (4.50 to 5.61) 5.21 (4.50 to 5.60) ER +ve 202 (54.0%) 158 (54.9%) ER -ve 119 (31.8%) 96 (33.3%) ER Unk 53 (14.2%) 34 (11.8%) BODY.TIMP1 IMMUNOREACTIVITY: Successful TIMP-1 staining was achieved for 291/321 cases. Three cases were lost to follow-up and excluded from subsequent analysis. Some TIMP-1 positive tumors displayed a homogenous staining, while others presented with a heterogenous staining pattern (Figure 1). Of the 288 cases available for analysis: 42% of the tumors had one or two TIMP-1 positive cores while 58% presented with two TIMP-1 negative cores. Figure 1Illustrative figures of TIMP1 staining. Panel A: Homogenous staining. Panel B: Heterogeneous staining. HER2, TOP2A and Ki67 Results for HER2 and TOP2A gene copy analyses and Ki67 protein analysis have been previously presented and are summarized in Table 2. Table 2HER2 and TOP2A gene status and Ki67 protein expression in the BR9601 cohort used for this study Amplified Deleted Normal HER2 FISH 274/288 (95.1%) 62 (22.6%) 212 (77.4%) TOP2A FISH 274/288 (95.1%) 25 (9.1%) 48 (17.5%) 201 (73.4%) High Low Ki67 284/288 (98.6%) 120 (42.3%) 164 (57.7%) BODY.ASSOCIATIONS BETWEEN TIMP-1 AND KI67 IMMUNOREACTIVITY AND :HER2 and TOP2A gene copy numbers and RFS and OS The prognostic significance of each of the biological markers included in this study was first tested on the entire included patient cohort (n = 288), irrespective of their allocated adjuvant chemotherapy. Exploratory analysis of these markers (minus TIMP-1) showed the results to be similar in this cohort and the 321 patients included in the original biomarker study [7]. TIMP-1 immunoreactivity was not significantly associated with RFS (HR = 0.83; 95% CI: 0.55 to 1.24; P = 0.36) or OS (HR = 0.69; 95% CI: 0.43 to 1.09; P = 0.11). However, TIMP-1 positivity was significantly associated with an increased number of positive lymph nodes (P = 0.01), ERα positive disease (P = 0.004), and decreased proliferation (measured by Ki67; P = 0.004; Table 3). Table 3Associations between TIMP-1, Ki-67, HER2 and TOP2A TIMP-1 Negative TIMP-1 Positive P-value Ki67 Low 83 (50.6%) 81 (67.5%) 0.004 High 81 (49.4%) 39 (32.5%) HER2 Normal* 119 (75.8%) 93 (79.5%) 0.470 Amplified 38 (24.2%) 24 (20.5%) TOP2A Deleted 32 (20.4%) 16 (13.7%) 0.310 Normal 110 (70.1%) 91 (77.8%) Amplified 15 (9.6%) 10 (8.5%) Normal includes samples with HER2 deletions and HER2 normal samples. Tumors were classified as 2T responsive (TOP2A abnormal and/or TIMP-1 negative) and 2T non-responsive (TOP2A normal and TIMP-1 immunoreactive) [13]. Using this definition, 183/274 (66.8%) of tumors were classed as 2T responsive and 91/274 (33.2%) as 2T non-responsive. There was no significant difference in RFS (HR = 0.91; 95% CI: 0.585 to 1.417; P = 0.68) and OS (HR = 0.773; 95% CI: 0.470 to 1.271; P = 0.30) between patients with the 2T responsive profile when compared to those with the non-responsive profile. A 2T responsive profile was associated with ERα negative disease (P = 0.0001), increased pathological grade (P = 0.005), and increased proliferation (P = 0.001). On the basis of TIMP-1 immunoreactivity and HER2 gene copy numbers, 181 (66%) of tumors were classified as HT responsive (HER2 amplified and/or TIMP-1 negative) and 93 (34%) as HT non-responsive (HER2 negative and TIMP-1 immunoreactive). Patients who had an HT responsive profile had a significantly decreased RFS (HR = 1.64; 95% CI: 1.035 to 2.6; P = 0.037) and OS (HR: 2.08; 95% CI: 1.21 to 3.56; P = 0.007) when compared to those with a non-responsive profile. A HT responsive profile was associated with ERα negative disease (P = 0.0002), increased pathological grade (P = 0.003), and increased proliferation (P = 0.0002). BODY.TREATMENT BY MARKER ANALYSIS OF THE INFLUENCE OF TIMP-1, :TOP2A and HER2 on RFS and OS benefits of E-CMF over CMF Subsequent analyses focused on possible differential effects of the expression of these markers on RFS and OS between patients receiving E-CMF and those treated with CMF alone. The results for HER2 and TOP2A gene copy number counts have been published previously [7]. Hazard ratios for TIMP-1 immunoreactivity and associated profiles between patients receiving E-CMF and CMF alone are summarized in Table 4. Treatment by marker (TxM) HRs demonstrates that there is no evidence that lack of TIMP-1 immunoreactivity alone or in combination with TOP2A or HER2 gene aberrations is predictive of anthracycline benefit. In patients with TIMP-1 immunoreactivity there is a trend towards an increase in RFS (HR = 0.48; 95% CI: 0.24 to 0.93; Figure 2A) and OS (HR = 0.46; 95% CI: 0.21 to 1.00) in patients treated with E-CMF compared to CMF alone. However, a similar trend for RFS (HR = 0.64; 95% CI: 0.38 to 1.10; Figure 2B) and OS (HR = 0.78; 95% CI: 0.45 to 1.38) was apparent in patients whose tumors were negative for TIMP-1 suggesting the trend is associated with benefit from treatment irrespective of the immunoreactivity of TIMP-1. Table 4Unadjusted HR estimates of the treatment by marker effects RFS OS HR 95% CI TxM (HR) HR 95% CI TxM (HR) TIMP-1 Negative (n = 167; 58%) 0.642 0.377 to 1.091 1.311 0.784 0.447 to 1.376 1.667 Positive (n = 121; 42%) 0.478 0.247 to 0.927 (0.562 to 3.056) 0.461 0.213 to 1.002 (0.641 to 4.339) 2T Profile Responsive (n = 183; 66.8%) 0.613 0.365 to 1.029 1.022 0.450 0.188 to 1.080 1.579 Non-responsive (n = 91; 33.2%) 0.582 0.277 to 1.221 (0.415 to 2.520) 0.739 0.428 to 1.277 (0.565 to 4.411) HT Profile Responsive (n = 181; 66.1%) 0.658 0.400 to 1.082 0.902 0.783 0.464 to 1.320 0.577 Non-responsive (n = 93; 33.9%) 0.531 0.235 to 1.200 (0.347 to 2.346) 0.384 0.141 to 1.046 (0.186 to 1.788) Figure 2Relapse free survival for E-CMF (solid line) versus CMF (dashed line). Panel A: TIMP-1 negative tumors. Panel B: TIMP-1 immunoreactive tumors. BODY.DISCUSSION: This study, which included 288/374 of the patients enrolled in the original BR9601 adjuvant study [2], failed to confirm the predictive value of TIMP-1 protein measurements for anthracycline benefit [10,13] either as a stand-alone biomarker or in combination with HER2 or TOP2A gene aberrations. It should be mentioned, however, that in the original biomarker study of the BR9601, neither HER2 nor TOP2A gene aberrations were associated with benefit from anthracyclines [7]. Although only 288 of the original 374 patient samples were available for this study, survival analyses confirmed the benefit from the addition of an anthracycline to CMF versus CMF alone in this subset of patients. Moreover, when analyzing the associations between patient outcome and HER2 and TOP2A gene copy number and Ki-67 protein in the 288 patients, similar results were obtained as reported previously [7]. It thus appears that these 288 patients are representative for the initial study population. The TIMP-1 immunostaining was performed using a validated anti-TIMP-1 monoclonal antibody and a strict protocol [18] and the scoring of TIMP-1 positivity/negativity was performed as previously described [10]. However, among the 288 patients 42% were found positive for TIMP-1, which is much less than what has previously been reported: in the DBCG 89D patient cohort 75% of the cases were TIMP-1 positive [10] and a similar distribution between TIMP-1 positivity/negativity was seen in the MA5 study [14]. While TIMP-1 immunoreactivity is often heterogeneous [10] and smaller TMA might impact on results, the cores used here were similar to those used in the MA5 study. Also while in the DBCG 89D study [10], the cores were taken from the invasive front of the tumors while in the BR9601 and MA5 study sampling was focused on tumor rich areas. TIMP-1 immunoreactivity was significantly associated with lower Ki67 immunoreactivity, suggesting that TIMP-1 positive tumors have a lower rate of proliferation, which in turn might result in reduced sensitivity to chemotherapy. Conversely, in the present study in which TIMP-1 positive tumors had a non-significant increased benefit from the E-CMF combination as compared to CMF alone is not explained by increased proliferation o An alternative explanation for the discordant results between the present study and our previous studies [10,13,14] is that the interaction depends on the regimens and/or the patient populations studied. The anthracycline regimens in the DBCG and the BR9601 studies are not identical, with a greater total dose and duration of epirubicin treatment in the DBCG study [3] as compared to BR9601 [2]. Furthermore, the DBCG trial recruited only pre-menopausal node-positive women, and patients did not receive adjuvant endocrine therapy. Conversely, in BR9601, both pre- and post-menopausal women were recruited, including 15% node negative tumors, and all ER positive cases were to receive five years of tamoxifen. Another noteworthy disparity between the trials was the higher percentage of ER positive patients recruited into BR9601 (54.9%) compared to the DBCG trial (33.7%). While these differences might explain the discordant results, there is no obvious a priori explanation as to why they should. BODY.CONCLUSIONS: In conclusion, this validation study of TIMP-1 breast cancer cell immunoreactivity as a predictive biomarker for adjuvant anthracycline benefit did not support the use of this marker to select patients for anthracycline treatment. Moreover, this study could not confirm any additive predictive value by combining TIMP-1 immunoreactivity with results on HER2 and TOP2A FISH analyses. BODY.ABBREVIATIONS: CEF: Cyclophosphamide, epirubicin, 5-fluorouracil; CEP17: Centromeric enumeration probe for chromosome 17; CMF: Cyclophosphamide, methotrexate, 5-fluorouracil; DFS: Disease free survival; E-CMF: Epirubicin-CMF; ER: Estrogen receptor; FISH: Fluorescent in situ hybridization; HR: Hazard ratio; IHC: Immunohistochemistry; i.v.: Intravenous; OS: Overall survival; RFS: Relapse free survival; TIIα: Topoisomerase IIα; TIMP-1: Tissue inhibitor of metalloproteinase 1; TMA: Tissue microarray. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: JB, AM, DC, CT and NB designed the study. JB, DC, CT and AM collected the tissue samples, established the database and performed the statistical analysis. EB and AB performed and scored the TIMP-1 immunhistochemistry. AM, JB and NB interpreted the data and drafted the manuscript. All authors have read and approved the manuscript for publication.

TITLE: Extravascular lung water assessed by transpulmonary single thermodilution and postmortem gravimetry in sheep ABSTRACT.INTRODUCTION: Acute lung injury is associated with accumulation of extravascular lung water (EVLW). The aim of the present study was to compare two methods for quantification of EVLW: transpulmonary single thermodilution (EVLWST) and postmortem gravimetric (EVLWG). ABSTRACT.METHODS: Eighteen instrumented and awake sheep were randomly assigned to one of three groups. All groups received Ringer's lactate (5 ml/kg per hour intravenously). To induce lung injury of different severities, sheep received Escherichia coli lipopolysaccharide 15 ng/kg per min intravenously for 6 hours (n = 7) or oleic acid 0.06 ml/kg intravenously over 30 min (n = 7). A third group (n = 4) was subjected to sham operation. Haemodynamic variables, including EVLWST, were measured using a PiCCOplus monitor (Pulsion Medical Systems, Munich, Germany), and the last measurement of EVLWST was compared with EVLWG. ABSTRACT.RESULTS: At the end of experiment, values for EVLWST (mean ± standard error) were 8.9 ± 0.6, 11.8 ± 1.0 and 18.2 ± 0.9 ml/kg in the sham-operated, lipopolysaccharide and oleic acid groups, respectively (P < 0.05). The corresponding values for EVLWIG were 6.2 ± 0.3, 7.1 ± 0.6 and 11.8 ± 0.7 ml/kg (P < 0.05). Ranges of EVLWIST and EVLWIG values were 7.5–21.0 and 4.9–14.5 ml/kg. Regression analysis between in vivo EVLWST and postmortem EVLWG yielded the following relation: EVLWST = 1.30 × EVLWG + 2.32 (n = 18, r = 0.85, P < 0.0001). The mean bias ± 2 standard deviations between EVLWST and EVLWG was 4.9 ± 5.1 ml/kg (P < 0.001). ABSTRACT.CONCLUSION: In sheep, EVLW determined using transpulmonary single thermodilution correlates closely with gravimetric measurements over a wide range of changes. However, transpulmonary single thermodilution overestimates EVLW as compared with postmortem gravimetry. BODY.INTRODUCTION: Acute lung injury (ALI) of septic and non-septic origin is a frequent cause of mortality in critically ill patients. During ALI, the inflammatory process in the lungs may increase the microvascular pressure and permeability, resulting in an accumulation of extravascular lung water (EVLW) and development of pulmonary oedema [1]. However, it is difficult to estimate the amount of oedema fluid at the bedside. Clinical examination, chest radiography and blood gases have proven to be of limited value in quantifying pulmonary oedema [1-3]. Several techniques to assess EVLW have therefore been developed. Among the various methods for measurement of EVLW, thermo-dye dilution has been used most frequently [4-8]. In animal models of lung oedema, this method has been evaluated by comparison with postmortem gravimetry, which is supposed to be the 'gold standard' of EVLW measurements [7-9]. In critically ill patients, fluid management guided by thermo-dye measured EVLW was associated with improved clinical outcome [10]. Hence, EVLW has been suggested to play a role as an independent predictor of the prognosis and course of illness [6,8,10]. However, the thermo-dye dilution method is relatively time consuming, cumbersome and expensive. For these reasons, the method has not gained general acceptance [4,5,7]. Use of a technique based on injection of a single thermo-indicator that can be detected using an indwelling arterial catheter was an appealing concept. Recent experimental and clinical studies have shown that EVLW assessed by single thermodilution (ST) exhibits good reproducibility and close agreement with the thermo-dye double indicator technique [11,12]. The ST method is simpler to apply, less invasive and more cost effective; all of these factors make it more suitable for use at the bedside. However, to date, this new method has been sparsely evaluated against gravimetry [13,14], and further validation is needed. Thus, the aim of the present study was to evaluate the accuracy of the ST technique by comparing it with that of postmortem gravimetry (EVLWG) in conscious sheep, in which ALI was induced either by lipopolysaccharide (LPS) or by oleic acid (OA). Both of these models of ALI are reproducible and have been extensively described [7,9,11,15,16]. BODY.METHODS: BODY.SURGICAL PREPARATION AND MEASUREMENTS: The study was approved by the Norwegian Experimental Animal Board and conducted in compliance with the European Convention on Animal Care. Eighteen yearling sheep weighing 27.5 ± 0.4 kg were instrumented, as a modification to previously described techniques [16-19], by inserting introducers into the left external jugular vein and common carotid artery. After 1–4 days of recovery, sheep were placed in an experimental pen. A thermodilution catheter (131HF7; Edwards Life Sciences, Irvine, CA, USA) was introduced into the pulmonary artery and a 4-Fr thermistor-tipped catheter (PV2014L16; Pulsion Medical Systems, Munich, Germany) into the carotid artery. The catheters were connected to pressure transducers (Transpac®III [Abbott, North Chicago, IL, USA] and PV8115 [Pulsion Medical Systems], respectively). Mean pulmonary arterial pressure (PAP), pulmonary arterial occlusion pressure (PAOP) and right atrial pressure (RAP) were displayed on a 565A Patient Data Monitor (Kone, Espoo, Finland) and recorded on a Gould Polygraph (Gould Instruments, Cleveland, OH, USA). Heart rate, mean systemic arterial pressure, cardiac index (CI), systemic vascular resistance index, extravascular lung water index (EVLWI) assessed using the single thermodilution technique (EVLWIST), pulmonary vascular permeability index (PVPI), global end-diastolic volume (GEDV) index (GEDVI), intrathoracic blood volume (ITBV) index (ITBVI) and blood temperature were determined at 1-hour intervals using a PiCCOplus monitor (Pulsion Medical Systems). Every value reported here is the mean of three consecutive measurements, each consisting of a 10 ml bolus of ice-cold 5% dextrose injected into the right atrium randomly during the respiratory cycle. To estimate EVLW we used the following formula [12]: EVLWST (ml) = ITTV - ITBV (where ITTV is the intrathoracic thermal volume). During clinical application of ST by means of the PiCCO monitor, ITBV is calculated as 1.25 × GEDV, the coefficient 1.25 being derived from critically ill patients [12]. However, in our previous investigations in sheep [17-19], in which ITBV was measured directly using the thermal-dye dilution technique, we found the coefficient to be 1.34 [14]. Thus, in the present study we used the corrected values of ITBVI, EVLWIST and PVPI, based on the following equation: ITBVI = 1.34 × GEDVI. Blood samples were drawn from the systemic arterial (a) and pulmonary arterial (v) lines and analyzed every two hours for blood gases and haemoglobin (Rapid 860; Chiron Diagnostics Corporation, East Walpole, MA, USA). The pulmonary vascular resistance index (PVRI), venous admixture (Qs/Qt), oxygen delivery index (DO2I) and oxygen consumption index were calculated as described previously [16,19,20]. BODY.EXPERIMENTAL PROTOCOL: After establishing a stable baseline at time 0 hours, awake and spontaneously breathing sheep were randomly assigned to three experimental groups: a sham operated group (n = 4); a LPS group (n = 7), receiving an intravenous infusion of Escherichia coli O26:B6 LPS (Sigma Chemical, St. Louis, MO, USA) at 15 ng/kg per min for 6 hours; and an OA group (n = 7), in which sheep were subjected to an intravenous infusion of OA (Sigma Chemical) 0.06 ml/kg mixed with the animal's blood. The duration of the infusion of OA was 30 min. During the experiment, all animals received a continuous infusion (5 ml/kg per hour) of Ringer's lactate, aiming to maintain intravascular volume at baseline levels. After the last measurements, at 2 hours in the OA group and at 6 hours in the sham-operated and the LPS groups, the sheep were anaesthetized and killed with a lethal dose of potassium chloride. Then, postmortem EVLWI (EVLWIG) was determined by gravimetry, as previously described [21-24]. BODY.STATISTICAL ANALYSIS: For each continuous variable, normality was checked using the Kholmogorov-Smirnov test. Data are expressed as mean ± standard error of the mean, and assessed by analysis of variance followed by Scheffe's test or test of contrasts, when appropriate. To evaluate the relationship between EVLWIST and EVLWIG, we used linear regression and Bland-Altman analysis. P < 0.05 was considered statistically significant. BODY.RESULTS: All animals survived until the end of the experiments. At baseline no significant differences were found between groups, as shown in Figs 1 and 2, and Tables 1 and 2. In the sham-operated sheep, all variables remained unchanged throughout the study. BODY.HAEMODYNAMIC AND EXTRAVASCULAR LUNG WATER MEASUREMENTS: Figure 1 and Table 1 show that LPS and OA induced marked increments in PAP and PVRI, peaking at 1 hour and subsequently decreasing gradually to values significantly above the respective baselines and the corresponding values in the sham-operated group. PAOP and RAP also rose in both the LPS and the OA groups (P < 0.05; data not shown). In parallel, LPS increased EVLWIST transiently by 20–35% (P < 0.05; Fig. 1). After OA administration, EVLWIST rose to a maximum of 84% above baseline (P < 0.01). At the end of the experiment, EVLWIST in the OA group had increased by 6.4 ml/kg and 9.3 ml/kg relative to the LPS and the sham-operated groups, corresponding to increments of 54% and 104%, respectively (P < 0.05). PVPI increased by 40% after LPS administration and by 90% after OA (P < 0.05; Fig. 1). GEDVI and ITBVI varied within 10–15% of baseline with no intergroup differences. As shown in Table 1, LPS caused tachycardia and a rise in CI accompanied by a slight increase in mean arterial pressure whereas systemic vascular resistance index decreased (P < 0.05). In contrast, in the OA group CI declined and systemic vascular resistance index increased relative to baseline (P < 0.05). BODY.OXYGENATION AND GAS EXCHANGE: LPS caused significant increments in mixed venous oxygen saturation, DO2I and Qs/Qt (Fig. 2). OA decreased both arterial and venous oxygenation and reduced DO2I (P < 0.05). Oxygen consumption index did not change significantly (not shown). LPS caused a transient reduction in arterial carbon dioxide tension and a rise in pH (P < 0.05; Table 2). After OA, pH decreased (P = 0.04). The haemoglobin concentration as well as the body temperature rose only in the LPS group (P < 0.05). BODY.LINEAR REGRESSION AND BLAND-ALTMAN ANALYSIS: As shown in Fig. 3, the regression analysis between EVLWST and postmortem EVLWG yielded the following relation: EVLWIST = 1.30 × EVLWG + 2.32 (n = 18, r = 0.85, P < 0.0001). Notably, the mean EVLWIST at the end of experiment was higher than EVLWIG: 13.6 ± 1.1 ml/kg versus 8.7 ± 0.7 ml/kg (P = 0.0005). Ranges of EVLWIST and EVLWIG values were 7.5–21.0 ml/kg and 4.9–14.5 ml/kg. According to the Bland-Altman analysis, the mean difference between EVLWIST and EVLWIG was 4.91 ml/kg, with upper and lower limits of agreement (± 2 standard deviations) of +9.99 ml/kg and -0.17 ml/kg, respectively (Fig. 4). The difference between methods increased with increasing values of mean EVLWI (n = 18, r = 0.64; P = 0.005); the regression line equation was as follows: EVLWIST - EVLWIG = 0.89 × ([EVLWIST + EVLWIG]/2) + 6.82. BODY.POSTMORTEM GRAVIMETRY: As shown in Fig. 5, EVLWIG in the OA group increased by 4.7 ml/kg and 5.6 ml/kg relative to the LPS and the sham-operated groups, amounting to increments by 65% and 90%, respectively (P = 0.001). BODY.DISCUSSION: The present findings confirm that, in sheep, EVLW measured using the single transpulmonary thermodilution technique correlates closely with EVLW determined using postmortem gravimetry. However, EVLWIST overestimates EVLWIG, with the degree of overestimation increasing with the severity of ALI. A number of experimental and clinical studies focused on the potential role of EVLW as a guide to diagnosis and treatment of critically ill patients [3,6-14,25,26]. During pulmonary oedema, accumulation of EVLW occurs before any changes take place in blood gases, chest radiogram and, ultimately, pressure variables. In addition, the latter variables are nonspecific diagnostic tools that are influenced by a variety of factors [2,4,5,8]. Thus, Boussat and coworkers [3] recently demonstrated that, in sepsis induced ALI, commonly used filling pressures such as PAOP and RAP are poor indicators of pulmonary oedema. Rather than those measures, they recommended direct measurement of EVLW. Consistent with this, we found that EVLW, in contrast to RAP, correlates with markers of lung injury in human septic shock [26]. Victims of ALI, regardless of pathogenesis, have a significantly higher EVLW than do other patients [6,26]. Hence, measurement of EVLW supports the diagnosis and may even improve clinical outcomes when used cautiously in combination with treatment protocols that are known to hasten the resolution of pulmonary oedema [10,25]. Instrumented awake sheep represents a stable experimental model for measuring cardiopulmonary variables, as demonstrated in the sham-operated group in the present study as well as by other investigators [15,27]. The model can be used to assess different interventions during ALI. Consistent with previous investigators [15,17,27], we observed that infusion of LPS and OA caused pulmonary hypertension, increased EVLW and impaired gas exchange. Despite increments in PAP, PAOP and PVRI, both ITBV and GEDV remained constant whereas PVPI (an index of microvascular permeability, calculated as the ratio of EVLW to pulmonary blood volume) increased significantly. Thus, the haemodynamic responses to LPS and OA are not purely hydrostatic but may also manifest as noncardiogenic permeability pulmonary oedema [13,15-18,27,28]. In the present study lung oedema was significantly more severe in the OA group than in the LPS group, which is consistent with the findings of other investigators [29]. In fact, OA causes acute haemorrhagic alveolitis, which may lead to acute endothelial and alveolar necrosis and a severe proteinaceous oedema [30]. In contrast, the LPS-induced ALI is initiated by accumulation of granulocytes and lymphocytes in the pulmonary microcirculation that results in more moderate damage to endothelial cells and lung oedema [31]. Lung injury in the LPS group was accompanied by a hyperdynamic circulatory state, which was manifested by systemic vasodilation and increments in CI and DO2I toward the end of the experiment. In contrast, in the OA group we observed cardiac depression and systemic vasoconstriction. This is consistent with previous investigations of LPS and OA [18,27,30,32]. Thus, ovine models exhibit a scatter of cardiopulmonary changes from normal in the sham-operated group to mild or moderate ALI in endotoxaemic sheep and moderate to severe ALI in animals subjected to OA. The significant correlation of EVLWIST and EVLWIG observed in the present study is consistent with findings of Katzenelson and coworkers [13], who validated EVLWIST versus postmortem gravimetry in dogs [13]. However, those investigators did not specifically assess the relationship between EVLWIST and EVLWIG in sepsis-induced ALI. In addition, their study was performed in anaesthetized and mechanically ventilated animals; hence, further investigation of the correlation in a conscious state was required. Recently, ST has been evaluated against the thermo-dye dilution method in both experimental and clinical settings [11,12]. The studies revealed a close agreement between the techniques. Thus, we believe that injection of cold saline can provide valuable information about the EVLW content and the severity of pulmonary oedema. During ALI, both ST and postmortem gravimetry demonstrated similar relative increases in EVLWI as compared with sham-operated animals. However, we noticed that ST overestimates the absolute values of EVLWI compared with the gravimetric technique – a discrepancy that increased with progression of pulmonary oedema. This finding could be accounted for by heat exchange of the thermal indicator with extravascular intrathoracic structures, such as the walls of the large vessels and the myocardium, and by recirculation of the indicator [8]. In addition, the coefficients for calculation of EVLWIST and ITBV may vary with weight and age, as well as between animal species [11]. Consequently, in the experimental setting EVLWIST requires a specific correction. In the present study we replaced the coefficient 1.25 used in humans in the ITBVI equation (i.e. ITBVI = 1.25 × GEDVI) with the recalculated 'ovine' coefficient 1.34 [14], which is based on 426 measurements in 48 animals [17-19]. In contrast to ST, the thermo-dye dilution technique runs the risk of underestimating EVLW in comparison with gravimetry [4]. This underestimation increases during ALI caused by instillation of hydrochloric acid into the airways, and has been explained by redistribution of pulmonary blood flow away from the oedematous areas. The redistribution is thought to prevent indicator diffusion and consequently to prevent detection of oedema [7]. In addition, detection of EVLW by thermo-dye dilution can be impaired by changes in CI as well as by positive end-expiratory pressure during mechanical ventilation [8,28]. Compared with other techniques for assessment of EVLW, ST may underestimate EVLW during pulmonary oedema due to intratracheal instillation of saline, although it is an accurate method in normal lungs [33]. However, intratracheal instillation of saline can also be criticized because a proportion of the fluid is rapidly absorbed and obscured from detection [34]. Notably, the use of postmortem gravimetry as the reference method for evaluating pulmonary oedema also has limitations [21,33]. For example, the method only allows one measurement and is therefore of no use in following variations over time. The application of gravimetry is limited almost exclusively to experimental studies. The comparison of gravimetric measurement with results of other techniques for determination of EVLW can be influenced by the duration from death to removal of the lungs and by pathophysiological changes in the lungs after cardiac arrest. Thus, the gravimetric technique can underestimate the real value of EVLWI because of partial reabsorption of fluid before excision of the lungs. BODY.CONCLUSION: The determination of EVLW by ST in sheep correlates closely with gravimetric measurements over a wide range of changes, and thus it may potentially be of benefit in quantifying lung oedema in critically ill patients. However, compared with postmortem gravimetry, single transpulmonary thermodilution overestimates the absolute values of EVLW. Thus, further studies are warranted to evaluate the accuracy of this method for managing ALI in humans. BODY.KEY MESSAGES: • In sheep, extravascular lung water assessed by transpulmonary single thermodilution correlates closely with gravimetric measurements over a wide range of changes. • Despite a moderate overestimation of the extravascular lung water content compared with post-mortem gravimetry, single thermodilution can be a useful tool for assessment of pulmonary oedema during ALI. BODY.COMPETING INTERESTS: This study was supported by Helse Nord (Norway), project number 4001.721.132; departmental funds, the Department of Anesthesiology, University Hospital of North Norway; and Pulsion Medical Systems (Germany). BODY.AUTHOR CONTRIBUTIONS: MYK participated in the design of study, performed statistical analysis, and drafted the manuscript. VVK participated in the design of study, performed statistical analysis, and prepared the figures. VVK and KW participated in the design of study. LJB participated in the design of study and provided coordination. All authors read and approved the final manuscript. BODY.ABBREVIATIONS: ALI = acute lung injury; CI = cardiac index; DO2I = oxygen delivery index; EVLW = extravascular lung water; EVLWI = extravascular lung water index; GEDV = global end-diastolic volume; GEDVI = global end-diastolic volume index; ITBV = intrathoracic blood volume; ITBVI = intrathoracic blood volume index; LPS = lipopolysaccharide; OA = oleic acid; PAOP = pulmonary arterial occlusion pressure; PAP = pulmonary arterial pressure; PVPI = pulmonary vascular permeability index; PVRI = pulmonary vascular resistance index; Qs/Qt = venous admixture; RAP = right atrial pressure; ST = single thermodilution.

TITLE: The Effect of Mulligan Mobilization Technique in Older Adults with Neck Pain: A Randomized Controlled, Double-Blind Study ABSTRACT.BACKGROUND: The purpose of this study was to examine the effect of Mulligan mobilization technique (MMT) on pain, range of motion (ROM), functional level, kinesiophobia, depression, and quality of life (QoL) in older adults with neck pain (NP). ABSTRACT.METHODS: Forty-two older adults with NP were included in the study, and they were randomly divided into two groups: traditional physiotherapy (TP) group and traditional physiotherapy-Mulligan mobilization (TPMM) group. Treatment program was scheduled for 10 sessions. Participants were assessed in terms of pain, ROM, functional level, kinesiophobia, depression, and QoL both pre- and posttreatment. ABSTRACT.RESULTS: Pain, ROM, functional level, kinesiophobia, depression, and QoL improved in both groups following treatment (p < 0.05). When comparing effects of these two treatment programs, it was observed that the TPMM group had a better outcome (p < 0.05) in terms of ROM, kinesiophobia, depression, and QoL. ABSTRACT.CONCLUSION: In older adults with NP, MMT has been found to have significant effects on pain, ROM, functional level, kinesiophobia, depression, and QoL as long as it is performed by a specialist. “This trial is registered with NCT03507907”. BODY.1. INTRODUCTION: Neck pain (NP) is one of the common musculoskeletal problems. NP can be caused by the stress over the musculoskeletal system due to postural disorders and may also be associated with other causes such as intervertebral disc herniation, nerve compression, or fracture [1]. The prevalence of NP is reported to range from 43% to 66.7%, which increases along with aging [2]. In a study conducted by March et al., on individuals over 65 years of age, the prevalence of NP was found to be 38.7% [3]. The use of various methods of manual treatments such as exercise, mobilization, and manipulation is supported by recent reviews on conservative treatments for mechanical NP [4]. Mulligan is one of the mobilization techniques that can be applied in case of NP. Being an important treatment tool used by most of the manual physical therapists, Mulligan mobilization techniques (MMTs) include several methods such as sustained natural epiphyseal glides (SNAGs) and natural epiphyseal glides that target the spine [5]. An immediate improvement in pain-free range of motion (ROM) in the involved joints is reposted as a result of applying this treatment approach [5, 6]. As a successful treatment approach for various orthopedics dysfunctions, a combination of the MMT concept along with several other methods of manual therapy has been suggested by the literature [7]. However, the application of the MMT for nonspecific NP in the older adults has not been investigated. When the literature is examined, there is no randomized controlled study investigating the effect of the MMT on older adults with NP. This study aims to investigate the effect of Mulligan mobilization technique on pain, range of motion, functional level, kinesiophobia, fear of movement, depression, and quality of life in older adults with neck pain. BODY.2. MATERIAL AND METHODS: BODY.2.1. STUDY DESIGN: This study was designed as a randomized controlled, double-blinded study. Patients who agreed to participate in the study were divided into two groups—as the traditional physiotherapy (TP) group and traditional physiotherapy-Mulligan mobilization (TPMM) group—using a matched randomization method based on gender and age. Both the researchers performing the assessment (CK) and the treatment (OB) and the participants were blind about the groups. All assessments were made by the same investigator (CK) before and after treatment. BODY.2.2. PARTICIPANTS: Individuals older than 65 years of age with NP, who were referred to the Physical Therapy and Rehabilitation Center of Ahi Evran University by a physiatrist (SS), were included in this study. Ongoing NP for at least 3 months having no neurological, rheumatological, or musculoskeletal problems and had not taken any analgesic medication for neck pain for the last 3 months were the inclusion criteria of the study. Exclusion criteria, however, were as follows: NP originating from various pathologies (tumor, rheumatoid arthritis, ankylosing spondylitis, fracture, dislocation, etc.), presence of cord compression, vertebrobasilar artery insufficiency, severe radiculopathy, osteoporosis or osteopenia (t score > −1), long-term use of anticoagulant or corticosteroid drugs, and patients who had received any treatment for their NP. In accordance with the guidelines approved by the local ethical committee and the Declaration of Human Rights, Helsinki, written informed consent was obtained from all participants. BODY.2.3. EVALUATION METHODS: BODY.2.3.1. DEMOGRAPHIC DATA: All patients were verbally inquired regarding their age, body mass index, and information about when the symptoms onset was. All these data were recorded. BODY.2.3.2. PAIN: The severity of pain at rest and during activity was assessed by visual analog scale (VAS). Participants were questioned about their average pain over the last 4 weeks. They were asked to mark the severity of their pain on a 10 cm long line, where 0 represented no pain and 10 stood for vicious pain [8]. The results were recorded in cm. BODY.2.3.3. NECK DISABILITY INDEX (NDI): This scale was used to evaluate how the participants' daily life was influenced by their NP. Total score of the scale ranges from 0 to 35, and higher scores indicate higher levels of disability [9]. BODY.2.3.4. TAMPA SCALE OF KINESIOPHOBIA (TSK): This scale was used to assess the patients' fear of pain or reinjury due to movement. It consists of 17 items and assesses various factors of fear/avoidance and injury/reinjury in several activities. Total score of the scale varies between 17 and 68, and higher scores represent higher levels of kinesiophobia [10]. BODY.2.3.5. RANGE OF MOTION: A universal goniometer was used to assess the ROM of the cervical vertebrae. Cervical flexion, extension, right and left lateral flexion, and right and left rotation movements were measured 3 times in an active manner while the patients were in a comfortable sitting position. The average value of the measurements was recorded as ROM [11]. The pain-free maximum degree of movement for each range was measured in degrees. This method has demonstrated good reliability [12]. BODY.2.3.6. BECK DEPRESSION INVENTORY (BDI): Participants' level of depression was assessed using BDI that consists of 21 categories with 4 options in each category. Each item has a score between 0 and 3, and total score varies from 0 to 63. Score ranges are interpreted as 0–9 points = minor depression, 10–16 points = mild depression, 17–29 points = moderate depression, and 30–63 points = severe depression [13]. BODY.2.3.7. SHORT FORM-36 (SF-36): This form was used to assess the QoL of the participants. This questionnaire consisted of 36 questions that are categorized into 8 groups as follows: physical role functioning, emotional role functioning, bodily pain, energy, social role functioning, mental health, and general health perception. Each category is scored on a 0–100 range, and higher scores indicate better QoL [14]. BODY.2.4. TREATMENT PROGRAMS: Forty-two older adults who agreed to participate in the study were divided into two groups using a matched randomization method. All participants in both the TP group and the TPMM group were included in a treatment program for 10 sessions. BODY.2.4.1. TRADITIONAL PHYSIOTHERAPY GROUP: In this study, traditional physiotherapy includes heat modalities, electrotherapy (transcutaneous electrical nerve stimulation (TENS) and ultrasound therapy), and exercises. Patients were asked to lie down in prone, and a pillow was placed under their abdomen for relaxation. We used the hot pack to induce vasodilitation and reduce muscle spasm in this study. A hot pack wrapped in 4 layers of towel was used for 15 minutes to treat for relaxing muscle spasms and for improving soft tissue elasticity [15]. TENS is a simple noninvasive modality and commonly used in both acute and chronic neck pains. The mechanism of analgesia with TENS is described as the “gate control theory” of pain, which is characterized by the modulation of nociceptive input in the dorsal horn of the spinal cord, by peripheral electrical stimulation of large sensory afferent nerves. Alternatively, electrical stimulation of certain receptor sites in the dorsal horn of the spinal cord may release endorphins and produce analgesia that can be reversed by the naloxone. A 50 Hz conventional TENS with a pulse duration <150 microseconds was used in our study. TENS was applied to the painful area of the neck for 20 minutes [16]. Ultrasound therapy, which is used to heat deep tissues, is one of the most important physical treatment methods. Ultrasound increases local metabolism, circulation, regeneration, and extensibility of connective tissue with its assuming thermal and mechanical effects. Ultrasound device (Chattanooga, USA) was used in the study. Ultrasound's gel was applied circularly with a thickness of 2-3 mm. Then, ultrasound with a 4 cm2 probe was applied with 1 MHz frequency and 1.5 Wt/cm2, for 5 min [17]. Furthermore, massage and exercise were suggested to participants. Classic regional massage was performed on the cervical and thoracic regions. Participants were informed and educated about effective ways of performing their daily life activities. In the context of therapeutic exercises, the older adults were trained for ROM exercises (anterior, lateral, and rotational) and posture exercises (shoulder circumduction, scapular adduction, and pectoral stretching). These exercises were repeated 5 times within the treatment program and 10 times after the program. BODY.2.4.2. TRADITIONAL PHYSIOTHERAPY-MULLIGAN MOBILIZATION GROUP: In this group, the MMT was applied in addition to the treatment program applied to the TP group. For two weeks, participants received SNAGs five days per week. According to the MMT, any minor positional fault at a joint can cause a limitation in its physiological movement. The first intervention of the MMT was the application of the natural apophyseal glides (NAGs) applied between C2 and C7. Patients were asked to sit and rest their back against a chair. The mobilization was reapplied by the oscillatory movements and was less than 6 repeats. SNAGs were a combination of mobilization and active movements for the vertebral column. Load-bearing positions were selected and performed at each spinal level. The technique was done without pain at the end of the joint movement [5]. With patients in a seated position, cervical SNAGs were applied with one thumb supported by the other that was placed—depending on the indication—on either the articular pillar or the spinous process of the upper vertebra of the functional spine unit. The therapist applied a passive intervertebral movement which was in a superoanterior direction along the facet plane. The therapist maintained this “glide” as the patient actively moved in any range of physiological movement and then sustained it at the end-range position for a few seconds. The release of the “glide” was when the patient returned to the starting position of the active movement [5]. For two weeks, this mobilization was repeated 6 times per session by a physiotherapist (OB) who holds a certificate in the MMT with 8 years of experience. BODY.2.5. SAMPLE SIZE: In accordance with the study by Ganesh et al., sample size was based on NDI scores in the patient with NP [4]. Their study was designed to investigate the effects of MMT on NP. Large effect size was calculated for this study. Therefore, with a statistically significant level of 5% (p=0.05), a statistical power of 80%, an effect size of 0.8, and a minimum of 21 participants were required per group. Allowing for a 10% dropout rate, 47 subjects were recruited into the study. BODY.2.6. STATISTICAL ANALYSIS: Statistical analyzes of the study were conducted using the “Statistical Package for Social Sciences” (SPSS) Version 18.0 (SPSS Inc. Chicago, IL, USA). Normal distribution of the data was examined using the “Shapiro–Wilk test.” All outcome analyses were conducted according to the intention-to-treat principle. The “Wilcoxon paired two sample test” was used to compare pretreatment and posttreatment intragroup differences in the findings obtained as a result of the evaluations. “Mann–Whitney U Test” was used to compare differences between the two groups. BODY.3. RESULTS: Among 47 older adults assessed at baseline, 3 were not meeting the inclusion criteria and 4 were lost to follow-up. Finally, the study was completed with 21 older adults in group TPMM, and 19 individuals in group TP (Figure 1). Sociodemographic data of the older adults in both the TP and TPMM groups were similar (p > 0.05) (Table 1). Comparing pretreatment and posttreatment findings indicated that the participants in both groups had a significant decrease in their pain, NDI, BDI, and TSK. They also had significant increase in their ROM and SF-36, except for the physical health condition category for the TPMM group (p < 0.05) (Table 2). Comparing the gains of the participants in the two groups indicated that pain, NDI, right/left neck rotation, left lateral flexion ROM, and mental health subcategory of SF-36 had similar improvement rate in both groups (p > 0.05) (Table 3). However, the two groups were different in terms of ROM (except for right/left neck rotation and left lateral flexion), TSK, BDI, and SF-36 (except for mental health subcategory), in all of which the TPMM group had greater improvements (Table 3). BODY.4. DISCUSSION: The results of this randomized, controlled, and double-blinded study showed that all participants had less pain, depression, and kinesiophobia; greater ROM; and better QoL and functional level. It was also found that there was greater improvement in joint ROM (except for right/left rotation and left lateral flexion), kinesiophobia, depression, and QoL (excluding mental health) in the TPMM group compared to the other group. As in all age groups, NP is a common health problem in the older adults [18]. As a result of the treatment programs of the present study with older adults, NP decreased in a similar way in both groups. In their study on individuals with chronic mechanical NP, Said et al. reported that the MMT had a greater impact on pain reduction compared to the traditional treatment [19]. According to the main explanation provided for the pain-reducing effect of the mobilization, mobilization movements correct positional faults in the bony structure and hence reduce pain [19, 20]. Some studies have reported that spinal manipulative therapy produces a specific hypoalgesic effect. Manipulation-induced hypoalgesia may seem to be nonopioid in nature; that is, it is not reversed by the naloxone and could not improve tolerance to repeated stimulation. It may occur concurrent to changes in sympathetic and motor systems. Furthermore, preliminary evidence indicates that mechanical hypoalgesia is more effective against thermal hypoalgesia in study populations. This specific effect is produced by manipulative therapy [21, 22]. It was believed that the precise mechanism of the sudden development brought about by SNAGs was complex containing many systems including sympathoexcitation and nonopioid hypoalgesia. [23]. El-Sayed et al. was emphasized that the rationale for the technique was initially based on a biomechanical explanation where repositioning of the superior articular facet using a SNAG would cause correction of positional fault, thus resulting in reduced pain and increased ROM in the neck [23]. In accordance with abovementioned studies, results of this study indicated a reduced level of pain in both groups. However, the fact that our participants consisted of older adults suffering NP was an outstanding point of the present study. One of the most common symptoms of cervical spine problems are restricted ROM [24]. According to the treatment results, there was an increase in the ROM in both groups. However, this increase was found to be greater in the TPMM group except for neck rotation and left lateral flexion. In their randomized controlled study, Gautam et al. divided 30 individuals with NP into 3 groups. They applied the MMT, Maitland technique, and TP to the first, second, and third groups, respectively. They reported that out of the three, the MMT had a greater impact on pain, ROM, and disability [11]. According to Edmonston and Singer, SNAGs are particularly important in painful movement dysfunctions as a result of degenerative changes, as these techniques make pain-free movements possible throughout the available ROM. Furthermore, the potential problems that may occur during passive movements are less likely as the patient is in control of the movement [25]. It is stated that in the MMT, zygapophyseal joints guide the spine, and thus applying NAGs and SNAGs lead to an increase in ROM [26]. The reason for the technique was based on a biomechanical explanation that repositioning the superior articular facet using SNAGs at the beginning would lead to correction of the positional impairment and thus result in pain reduction and increased ROM. Furthermore, normal movement on the articular surface is necessary to maintain the mobility of adjacent nerves that altered biomechanics may affect the nervous outgrowth. Because of this, restoration of normal mechanics in joint space may normalize negative neuron-names that appear as a consequence of limited joint movement [23]. Many studies have indicated a decrease in cervical joint mobility as a result of aging, as well [27–29]. Older adults with neck pain were included in our study, and the MMT were found to improve ROM of the joints. In the literature, however, the MMT seems to be applied to young adults [11, 30]. For this reason, there is a need for studies that investigate the efficacy of the MMT on older adults with NP. Ganesh et al. divided individuals aged 21–45 years with mechanical neck pain into 3 groups in their studies. They applied Mulligan mobilization to group 1, Maitland mobilization to group 2, and exercise therapy only to group 3. At the end of their studies, they found that manual therapy techniques were not as good as pain relief, increase to ROM and neck disability as compared to exercise (level of evidence = 1C) [4]. Shin and Lee designed a single blind and randomized controlled trial in their study and divided randomly forty patients with headache into the SNAGs group and the control group. Shin and Lee were reported that the SNAGs technique can help to relieve headache and cervical pain in middle-aged women suffering from cervical headache (level of evidence = 1B) [31]. El-Sayed et al. divided randomly patients with radiculopathy whose ages were 40–55 years into the SNAGs + conventional physical therapy group and the conventional physical therapy group in their study. They explained that the SNAGs technique combined with TP is more effective in the rehabilitation program (level of evidence = 1B) [23]. Copurgensli et al. designed a single blind and randomized controlled trial. They were randomly placed into three groups: group 1: conventional rehabilitation; group 2: conventional rehabilitation and MMT; and group 3: conventional rehabilitation and kinesio taping. Results of their study showed that the MMT and kinesio taping have no additional effects on neck pain, muscle strength, and neck-related disability. Furthermore, they said that the use of the MMT and kinesio taping in addition to conventional rehabilitation, the gain in cervical ROM, and deep cervical flexor muscle strength may be increased in patients with cervical spondylosis (level of evidence = 1B) [32]. These studies are generally randomized controlled (level of evidence = 1B) studies in the literature. In comparison studies of the MMT with other treatments there are different opinions about whether it is effective or not [4, 23, 31, 32]. In our study, older adults aged 65 years and over were included, and the MMT has been found to have significant effects on pain, ROM, functional level, kinesiophobia, depression, and QoL. Moreover, the studies in young adults in the literature have been designed nonblindness or single blindness. This study was designed as a double-blinded-randomized controlled trial. To the best of our knowledge, there were few studies which compare the effects of the MMT on pain, ROM, functional level, kinesiophobia, depression, and QoL in older adults with chronic neck pain. It has been shown that there is an important relationship between pain and kinesophobia in individuals suffering NP. As a result of our study, it was determined that fear of movement decreased in both groups. This decrease was more pronounced in the TPMM group. It is also stated that, in case of NP, ROM in the cervical region decreases, movements are slower than normal, and proprioception is impaired [24, 30]. It is thought that any increase in ROM results in an increase in the proprioceptive sensation in the neck region, which may result in reduced kinesophobia in patients. NDI was used in the present study to assess the patients' disabilities in daily life due to their NP. According to our posttreatment evaluations, NDI results had improved in both TP and TPMM groups in a similar manner. Our results are in agreement with Sudarshan, who applied a simultaneous combination of neurodynamic mobilization and SNAGs and reported immediate improvement in VAS, cervical ROM, and NDI. In our study, similar development was achieved in both groups [33]. This is thought to be due to the fact that older adults are able to perform their daily life activities better as a result of reduced pain. SF-36 was used to assess the QoL of the participants. This questionnaire was developed specifically to assess QoL in patients with physical illnesses [14]. At the end of our treatment programs, there was an increase in QoL in both the TP and TPMM groups, which was found to be higher in the TPMM group except for the mental health score. Maiers et al. investigated the effects of spinal manual therapy (cervical joint and soft tissue mobilization) and exercises in the older adults with chronic NP. They reported minor improvement in QoL following the treatment; however, this improvement was not statistically significant [18]. Even though the present study is similar to the one by Maiers et al. in terms of patient population, we achieved greater gains in QoL of our participants. The TPMM group showed more pronounced improvement in QoL (except for the mental health scores), and this is thought to be a result of higher ROM and reduced pain, both of which have positive effects on QoL. These two parameters are more significantly gained in the TPMM group. BDI was used to determine the risk of depression in patients and/or to measure the level of depressive symptoms and the change in its severity [13]. As a result of this study, both the TP and TPMM groups showed a decrease in depression levels, and this decrease was found to be higher in the TPMM group. The most important outcome of the present study is that the MMT can be safely applied in older adults with NP without harming the patients. In addition, functional limitations in older adults with NP were reduced, and pain-free ROM was obtained. Although there are some studies in the literature evaluating the efficacy of the MMT in individuals suffering NP, there are no studies investigating the efficacy of this technique on older adults. Two strengths of this study are that the patient group consists of older adults and that it is a random-controlled double-blind study. Long-term effects of the MMT in older adults with NP are not investigated, which is a limitation of the present study. To have more precise results, it is necessary to continue long-term follow-up evaluations of the patients to investigate the rate of recurrence in each group. BODY.5. CONCLUSION: According to the findings of this study, applying the MMT in older adults with NP has positive effects on pain, ROM, functional level, kinesiophobia, fear of movement, ES, and QoL.

TITLE: A Randomised, Placebo-Controlled, First-In-Human Study of a Novel Clade C Therapeutic Peptide Vaccine Administered Ex Vivo to Autologous White Blood Cells in HIV Infected IndividualsGag Peptide Vaccination in HIV Infected Patients ABSTRACT.BACKGROUND: Preclinical studies of overlapping 15mer peptides, spanning SIV, SHIV or HIV, pulsed on autologous PBMC ex vivo have demonstrated high level, virus-specific T cell responses and viral suppression in non-human primates (NHP). Opal-HIV-Gag(c) consists of 120 synthetic 15mer peptides spanning Clade C, consensus Gag, manufactured to current good manufacturing practice; having been evaluated in a good laboratory practice toxicology study in Macaca mulatta. We evaluated the safety and preliminary immunogenicity of such peptides administered intravenously after short-duration ex vivo incubation, to HIV-positive adults on suppressive antiretroviral therapy. ABSTRACT.METHODS AND FINDINGS: A first-in-human, placebo-controlled, double-blind, dose escalation study was conducted. Twenty-three patients with virus suppressed by antiretroviral therapy were enrolled in four groups 12 mg (n = 6), 24 mg (n = 6), 48 mg (n = 2) or matching placebo (n = 8). Treatment was administered intravenously after bedside enrichment of 120 mL whole blood for white cells using a closed system (Sepax S-100 device), with ex vivo peptide admixture (or diluent alone) and 37°C incubation for one hour prior to reinfusion. Patients received 4 administrations at monthly intervals followed by a 12-week observation post-treatment. Opal-HIV-Gag(c) was reasonably tolerated at doses of 12 and 24 mg. There was an increased incidence of temporally associated pyrexia, chills, and transient/self-limiting lymphopenia in Opal-HIV-Gag(c) recipients compared to placebo. The study was terminated early, after two patients were recruited to the 48 mg cohort; a serious adverse event of hypotension, tachycardia secondary to diarrhoea occurred following a single product administration. An infectious cause for the event could not be identified, leaving the possibility of immunologically mediated product reaction. ABSTRACT.CONCLUSIONS: A serious, potentially life-threatening event of hypotension led to early, precautionary termination of the study. In the absence of a clearly defined mechanism or ability to predict such occurrence, further development of Opal-HIV-Gag(c) will not be undertaken in the current form. ABSTRACT.REGISTRATION: ClinicalTrials.gov NCT01123915; EudraCT: 2008-005142-23 BODY.INTRODUCTION: HIV remains a significant global health problem, despite the availability of a range of antiretroviral treatments and strategies. An estimated 2.7 million people were newly infected with HIV and approximately 1.8 million died from HIV/AIDS in 2011 [1]. The use of combination highly active antiretroviral therapy (HAART) has significantly improved prospects for HIV infected individuals and has lowered transmission rates. However, the treatment regimens are complex, expensive and may be associated with treatment-limiting side effects and the emergence of drug resistant viral strains. These factors remain critical barriers to the management of HIV/AIDS, particularly in economically disadvantaged communities. The availability of an immunotherapy, which either delays the introduction of HAART or complements treatment by HAART, would be an important advance in treating HIV. The induction of HIV-specific T-cell responses is critical to effective control of viraemia and delaying subsequent progression to AIDS [2]–[4]. Cytotoxic T-lymphocyte responses to the HIV structural protein Gag have been consistently associated with low viral load [5], [6], with evidence that viral escape from Gag-specific T-cell responses occurs at the expense of viral fitness [5], [7]–[9]. This suggests that Gag-specific cellular immune responses may be an appropriate target antigen for an HIV therapeutic vaccine. Overlapping peptide-pulsed autologous lymphocytes (OPAL) is a novel immunotherapy being developed for the treatment of HIV. The therapy involves pulsing autologous peripheral blood mononuclear cells (PBMCs), enriched white blood cells (WBCs) or whole blood ex vivo, with a mixture of synthetic 15mer peptides overlapping by 11 amino acids. This approach, used in non-human primates, induced high-frequency, broad, polyfunctional CD4+ and CD8+ SIV-specific T cell responses [7]–[10] that resulted in a sustained, 10-fold reduction in SIV viral load in vaccinated animals [10], [11]. The vaccine for clinical evaluation, Opal-HIV-Gag(c), was manufactured to match the Gag clade C Durban consensus sequence [6]. The clade C subtype circulates in Southern Africa, India, and China, and is responsible for over 50% of all HIV infections worldwide [12]. Since Gag is highly conserved across clades, it was reasoned that Opal-HIV-Gag(c) would have broad cross-clade reactivity [11], [13]–[15]. This phase I study was the first step to determine whether the immunotherapy Opal-HIV-Gag(c) might have utility as a treatment for HIV when administered ex vivo to enriched WBCs. The aim was to evaluate the safety of the study vaccine, Opal-HIV-Gag(c) compared to placebo, at three dose concentrations. The secondary aim was to evaluate the immunogenicity in T-cells and to assess the impact of Opal-HIV-Gag(c) on HIV infection. The study was terminated early for safety reasons. BODY.METHODS: The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. BODY.ETHICS STATEMENT AND REGULATORY APPROVALS: The protocol, designated Opal-HIV-1001 and titled “A phase 1, dose escalating, single centre, double blind study of the safety and immunogenicity of Opal-HIV-Gag(c) in HIV-1 positive subjects” was sponsored by Medicines Development, Melbourne, Australia. The study was conducted in compliance with the International Conference on Harmonisation Good Clinical Practice, the Declaration of Helsinki, and was registered with EudraCT 2008-005142-23 prior to enrolment of participants. Ethical approval was granted by the The Royal Marsden Ethics Committee of the National Research Ethics Service, UK. All study participants voluntarily provided written informed consent before any study procedures were undertaken. BODY.OBJECTIVES: The primary objective was to assess the safety of Opal-HIV-Gag(c) at three dose concentrations compared to placebo in HIV-1 individuals receiving stable HAART, while the secondary objectives were to evaluate the immunogenicity and impact of Opal-HIV-Gag(c)) treatment on HIV-1 infection. BODY.STUDY DESIGN AND PARTICIPANTS: This was a phase I, first-in-human, double blind, placebo controlled, randomised, dose escalation study conducted at a single centre, Chelsea & Westminster Hospital, London, UK from May 2010 to October 2011. Participants were recruited from the ambulatory clinics at Chelsea & Westminster Hospital, London UK, Royal Berkshire Hospital, Reading UK and Northamptonshire General Hospital, Northampton, UK. After discussion and written confirmation of informed consent, eligibility was confirmed according to the following major criteria: aged 18–60 years, inclusive; HIV-1 infected; receiving stable antiretroviral therapy with at least 3 active drugs for a minimum of 2 months and with undetectable viral load for 6 months prior to planned study baseline; CD4+ T-cell counts >350 cells/mm3 with a nadir >100 cells/mm3, and a positive ex vivo or 10 day cultured IFNγ ELIspot assay to Opal-HIV-Gag(c) peptides. Exclusion criteria included: infection with hepatitis B or C; an AIDS defining condition within 42 days of Baseline; having received any immunomodulatory agents/vaccine within 60 days or any blood products within 6 months of Screening. The full inclusion/exclusion criteria for the study can be found in protocol S1. The study protocol planned to enrol a total of up to 27 patients in three sequentially completed, ascending-dose cohorts of 9 patients each. Within each cohort, patients were randomised to receive Opal-HIV-Gag(c) (n = 6) or placebo (DMSO, n = 3) and stratified by Clade. In addition, a sentinel cohort of one Opal-HIV-Gag(c) and one placebo patient were dosed prior to the balance of the cohort. The randomisation code was computer generated and prepared by an independent statistician. Participants received doses at 4 weekly intervals on Day zero and weeks 4, 8 and 12 followed by a 12 week post-treatment follow-up safety period. Dose escalation was permitted after all participants at each dose level completed at least 2 doses and the safety data were reviewed by the independent Data Safety Monitoring Board. Patients were monitored throughout the study for adverse events, concurrent medications, physical findings, vital signs, immunological markers, viral load, CD4+ T cell counts, and safety bloods. Clinical trial monitoring was performed by AptivSolutions, Hemel Hempstead, UK; biochemistry, haematology, viral load and T-cell enumeration was conducted by The Doctor’s Laboratory, London, UK; HLA genotyping was conducted the University of Oklahoma Health Sciences Centre, OK, USA; data management and statistical analysis for the study was performed by iNC Research, Oakleigh, Australia, and; immunogenicity was evaluated at Imperial College, London, UK and Oxford University, Oxford, UK. BODY.INTERVENTIONS AND VACCINES: Opal-HIV-Gag(c) consists of 120 peptides of 15 amino acids in length overlapping the preceding proceeding peptide by 11 amino acids. The 120 peptides span the HIV-1 Durban 2005 Clade C Gag protein consensus sequence and were manufactured to current Good Manufacturing Practice as defined by United States Part 21 Code of Federal Regulations by CS Bio, Inc. (San Mateo, CA). All 120 peptides were mixed in equal weight quantities, lyophilised and terminally gamma irradiated. A repeat dose Good Laboratory Practice toxicology study was completed in non-human primates prior to initiation of this study. Opal-HIV-Gag(c) was reconstituted prior to administration in dimethyl sulfoxide (DMSO) United States Pharmacopoea (WAK Chemie GmbH, Germany). DMSO at the identical concentration was used as the placebo. Opal-HIV-Gag(c) and diluent were stored frozen at −20°C±5°C in an entry restricted and temperature-monitored facility at the study site and thawed immediately prior to use, with doses prepared individually. Opal-HIV-Gag(c) or placebo were administered by drawing 120 mL of whole blood, enriching the white blood cells into a resultant 20 mL volume using a Sepax S-100 cell separation device (Biosafe SA, Switzerland) adding Opal-HIV-Gag(c) and were incubated at 37°C for one hour, prior to intravenous re-infusion. The concentration of DMSO in the reinfusion was 4% for both Opal-HIV-Gag(c)) or placebo. The clinical doses were selected based on the non-human primate efficacy and safety studies. In the repeat dose toxicology study in non-human primates, the maximum dose was 5 doses of 18.5 mg Opal-HIV-Gag(c), which is the equivalent of 74 mg/square metre (m2) of Body Surface Area (BSA) (assumes 0.25 m2 BSA for a monkey of 3 kilograms [kg]), or 6.2 mg/kg at each of the 5 administrations and this was also determined to be the no observable adverse effect level (NOAEL). The clinical starting dose was 12 mg or 7.4 mg/m2 (0.2 mg/kg, assuming 1.62 m2 BSA for a 60 kg human) at each of four administrations and the maximum dose proposed in the clinical trial was 48 mg or 29.6 mg/m2 (0.8 mg/kg). Hence, the first-in-human starting dose for the study was approximately 10 times lower than the NOAEL in non-human primates (based on the USA Food and Drug Administration Guidance for Industry and Reviewers Estimating the Safe Starting Dose in Clinical Trials for Therapeutics in Adult Healthy Volunteers [9]). Though the patients recruited to this study were infected with HIV-1, the eligibility criteria were suitably defined such that their health status (on stable antiretroviral therapy) could otherwise reasonably be classified as healthy adult participants. BODY.IMMUNOGENICITY: The induction of T-cell immunogenicity was assessed by using ex vivo ELISpot responses using Opal-HIV-Gag(c), Tat, Rev, Nef, a mock as a negative control and cytomegalovirus peptides to stimulate T-cells in accordance with the laboratories’ standard procedures. BODY.SAMPLE SIZE AND STATISTICAL DESIGN: The methodology for reporting study data was detailed in the Statistical Analysis Plan (SAP). Appropriate descriptive statistics for the data were determined using SAS (version 9.2 - SAS Institute Inc., Cary, North Carolina, USA). Adverse events were coded according to the Medical Dictionary for Regulatory Activities (MedDRA) (version 12.0). Concomitant medications were coded using the latest version (Quarter 2, 2010) of the World Health Organisation (WHO) Drug coding dictionary. All data compiled for participants prior to the point of discontinuation has been used for analyses with all withdrawals being included in analysis up to the time of withdrawal regardless of duration of treatment. No substitutions were made for missing data. All analyses were based on available data, unless otherwise stated. Blinded interim reviews of immunogenicity data (to W14) were conducted after the 12 mg and 24 mg Cohorts were completed. All statistical analyses were carried out using two sided tests at the 5% level of significance. In cases where the parameters did not follow a normal distribution, log transformations were used. If the log transformed data was not normally distributed, a non-parametric test (Kruskal-Wallis) was used to analyse the difference in population medians. The sample size for the study was selected based on industry guidance and Phase I study design [13]–[16] and, as such, no formal sample size calculation was performed for this study. BODY.RESULTS: BODY.STUDY POPULATION: Twenty three patients satisfied the inclusion and exclusion criteria and were randomised to receive 12 mg (n = 6), 24 mg (n = 6), 48 mg (n = 2) or placebo (n = 9, with only 8 being analysed after receiving intervention). Five participants withdrew from the study: one patient, allocated to receive placebo, due to equipment failure prior to treatment administration (this patient was replaced); 1 receiving 48 mg withdrew due to a serious adverse event (SAE) leading to early study termination; and three patients (n = 1 48 mg, n = 2 placebo) were required to withdraw when the study was terminated. In addition, one patient with elevated ALT due to concurrent therapy withdrew from treatment but remained on the study. Despite the small number of patients, the demographic characteristics of the cohorts were not markedly different (Table S1). BODY.IMPACT OF OPAL-HIV-GAG(C) ON HIV VIRAL LOAD AND ABSOLUTE CD4 COUNTS: HIV-1 viral load remained well controlled, without any result, confirmed by repeat, above 50 copies/ml for all study patients throughout the study period (not shown), indicating an absence of viral rebound. In all cohorts, CD4+ cell count varied around a stable plateau, consistent with the pattern of variability seen in normal clinical follow-up and with no temporal association with administration of Opal-HIV-Gag(c)) or placebo (data not shown). BODY.SAFETY ANALYSES: Opal-HIV-Gag(c) was generally well tolerated. The most commonly occurring AEs (ranked on total number of participants in all treatment cohorts experiencing AE) are presented in Table S2. At the 12 mg or 24 mg cohorts, there was no evidence of an increasing incidence or severity of adverse events with increasing dose and there was a similar number of events in patients receiving Opal-HIV-Gag(c) or placebo. Body temperature increases were temporally associated with Opal-HIV-Gag(c), regardless of dose, and there was an increased incidence of rigors, chills and transient lymphopenia temporally associated with Opal-HIV-Gag(c) but not placebo treatment. The second patient randomised to receive 48 mg Opal-HIV-Gag(c) experienced a treatment and study-terminating SAE. The event was comprised of hypotension, tachycardia, diarrhoea, and anuria. The patient, a 53-year-old male originally from Kenya, was infected with HIV-1 clade B/D virus since 2006 and had achieved virologic suppression with tenofovir, emtricitabine and efavirenz since shortly after diagnosis. Pre-treatment, he reported good health with no clinically relevant abnormalities detected on full physical examination or in baseline laboratory values. Within 2 hours of completion of the first infusion, he began to experience cramping, abdominal discomfort and subsequently passed large volume, watery bowel motions on 3 occasions within the ensuing hour, with simultaneous vomiting on two occasions. There was no blood present in either stool or vomit, and examination revealed a quiescent abdomen following evacuation with no evidence of rash or angioedema. Though afebrile during this period, the fluid losses resulted in a hypovolaemic state with a drop in systolic blood pressure, tachycardia and tachypnoea. No specific therapy was administered to manage any presumed cause of this event, with only intravenous fluid replacement and low molecular weight heparin to prevent venous thrombosis. The patient made a rapid and full recovery and was discharged from hospital on day 5 after administration of investigational product, with resolution of hypotension, tachycardia, diarrhoea and anuria. Stool cultures were negative for cryptosporidia, C. difficile, E.coli 0157, Salmonella, Shigella, Campylobacter. No ova, cysts or parasites were detected on microscopic examination and ELISA for viruses (adenovirus, norovirus and rotavirus) were negative. There was no growth from blood or urine cultures and no plasmodium were visible on blood film on examining for the presence of malaria. Both polymerase chain reaction (PCR) for rotavirus and norovirus on stool sample and C.perfringens enterotoxin test on stool were also negative. There was no rise from baseline to 24 hours post admission in mast cell tryptase thus reducing the likelihood of diagnosis of an anaphylactoid-type reaction. Lymphopenia was reported 4 hours post-dose, but had resolved to within normal reference range within 2 days. BODY.IMMUNOGENICITY: Comparisons between treatment groups for immunogenicity by ex vivo IFN-γ ELISpot showed no overall difference for any of the parameters tested (Opal-HIV-Gag(c), Mock, Rev, Tat and Nef) compared to Baseline or placebo. There was an apparent response in area under the curve (AUC) for Rev (p = 0.012 before correction for multiple tests) for the 12 mg Opal-HIV-Gag(c) group compared with pooled placebo participants (n = 8) (data not shown; these data are described more fully in a separate manuscript), but this was not significant after Bonferroni correction for multiple tests. Individually, there were two participants, one in each of the 12 mg and 24 mg Opal-HIV-Gag(c) cohorts, who responded at W14 (2 weeks post-treatment) compared to Baseline (data not shown; these data are described more fully in a separate manuscript). BODY.DISCUSSION: This was a first-in-human Phase I, double-blind, placebo-controlled, dose-escalation study of the safety and immunogenicity of Opal-HIV-Gag(c) at 3 dose levels in patients with well controlled HIV-1 infection. The initial clinical trial program planned for the Opal vaccine was in two initial steps. The current study was designed as a safety evaluation in patients with well-controlled viral replication on antiretroviral therapy and without an interruption to therapy. Subsequent to this study, it was intended to evaluate efficacy in a Phase I/II study in an adult and then in a paediatric population where efficacy could be established in a treatment interruption model. The study escalated through 12 mg and 24 mg before being terminated due to a SAE in the 48 mg cohort. Although the sentinel patient allocated to receive the active product had tolerated 48 mg without any notable side effects, the second patient’s hypotension and anuria secondary to diarrhoea and vomiting occurring approximately 2 hours after dosing led directly to Sponsor and Investigator-agreed clinical hold. The thorough investigation included the vaccine, administration method, and the patient’s recent and past medical history. An identifiable cause for the event in standard areas of evaluation (infectious agent/food poisoning, co-morbidities, medical cause other than the study product, study product not meeting specification and or the study procedures not being followed) could not be identified. The incidence of gastrointestinal findings in all other patients between Opal-HIV-Gag(c) and placebo recipients was similar, and there was no evidence of immunotoxicity in the GLP toxicology study conducted in Macaca mulatta. As a result of the failure to identify an alternative causative agent or to identify methods of ameliorating the event should it occur again in other patients, the study was terminated as a safety precaution. In all other patients, Opal-HIV-Gag(c) was well tolerated. Consistent with many phase I studies, headache was the most commonly reported AE in this study and occurred at similar rates in patients receiving either Opal-HIV-Gag(c) or placebo. This may be associated with the study requirements for fasting and caffeine withdrawal. Consistently observed, was an increased incidence of fever, rigor, headache and transient, self-limiting lymphopenia in patients receiving Opal-HIV-Gag(c) at any dose but not placebo recipients. The temporal association of these events with Opal-HIV-Gag(c) is consistent with an innate immune response and provides evidence of a biological response to the peptides rather than the ex vivo administration method or diluent [16], [17]. Adverse events frequently reported in the literature for DMSO (e.g. sedation, headache, facial flushing [17]–[19], nausea, vomiting, abdominal cramps, dizziness [18]–[20] and a taste of garlic or onion [20]–[22] were not observed in this study. Ex vivo white blood cell enrichment conducted bed-side in the closed system Biosafe Sepax S-100 device was employed as a more practical alternative to a laboratory based PBMC separation methodology. Prior to the conduct of this study, a separate pilot study was conducted in 6 patients to evaluate the equipment. In clinical use, the equipment failed on a number of occasions, limiting its potential for use as a real-time tool for WBC enrichment. The translation from animal models to humans remains problematic in HIV vaccinology with this study non-predictive for immunogenicity, safety and efficacy between non-human primates and humans [21], [22]. The absence of a clear immunogenic signal in this study is in marked contrast to the significant T-cell immunogenicity observed in studies in Macaca nemestrina with the Opal vaccination methodology [1], [10]. A GLP, repeat dose, non-human primate (Macaca mulatta) toxicology study was conducted to evaluate the safety and immunotoxicity of Opal HIV Gag(c). Eighteen animals were randomly allocated to receive 1.85 or 18.5 milligram (mg) of cGMP Opal HIV Gag(c) in DMSO or DMSO only (n = 6 per group). Treatment was added to whole blood ex vivo, incubated for 1 hour at 37 degrees (°) Celsius (C), and reinfused on 5 separate occasions. There were no clinically relevant adverse findings in either Opal HIV Gag(c) or control animals with all animals remaining healthy throughout the study. There were no treatment related changes in haematology, serum chemistries or urinalysis, and no relevant histological findings upon necropsy. The exposure to peptides, and the number of cells exposed to peptides ex vivo were both within the range or greater than that used in the Macaca nemestrina studies (0.035 to 0.07 mg peptides per million WBC in non-human primates compared to 0.018 to 0.285 mg peptides per million WBC in this study). In determining the adequacy of the clinical dose for immunogenicity purposes, both the amount of Opal HIV Gag(c) and the number of PBMCs exposed have been taken into account. Allometric scaling on body surface area (BSA) was performed to determine the blood volume required in the clinic to yield a proportionally similar number of PBMCs to that shown to be effective in macaques. The blood volume ranged from 29.5 mL to 120 mL, and thus 120 mL of venous blood was drawn from each patient. From the non-human primate non-clinical studies, it was estimated that 9×106 to 1.8×107 PBMCs were exposed to peptides at each administration for each macaque. Allometrically scaling this to humans (based on BSA) yielded an ideal PBMC count of 5.9×107 to 1.2×108 for Opal treatment per person, the range achieved in the clinical trial. Finally, a range of 0.035 mg to 0.07 mg of peptide per million PBMCs was shown to be efficacious in the non-human primate model. Extrapolating this to humans with the expected PBMC yield from 120 mL of whole blood requires a range of minimum doses of 4.2 to 16.8 mg per administration. The clinical doses evaluated span this range with the minimum dose of 12 mg and the maximum dose of 48 mg. In conclusion, in this double blind, placebo controlled, dose escalation study, Opal-HIV-Gag(c)) was generally well tolerated in adults with well-controlled HIV-1 infection at doses of 12 and 24 mg. There was an increased incidence of pyrexia, chills, rigor, and transient (and self-limiting) lymphopaenia in Opal-HIV-Gag(c) recipients compared to placebo. There were no clear differences in dose on the incidence of laboratory abnormalities or the nature, incidence or severity of adverse events. There was no evidence of a treatment effect on T-cell responses, measured by ex vivo ELISpot, after administration of Opal-HIV-Gag(c).). An SAE of life-threatening hypotension at a dose of 48 mg lead to early, precautionary termination of the study. Further development of OPAL will not be undertaken in the current form. BODY.SUPPORTING INFORMATION: Table S1 Patient characteristics. (DOCX) Click here for additional data file. Table S2 Summary of common adverse events. (DOCX) Click here for additional data file. Protocol S1 Trial protocol. (PDF) Click here for additional data file. Diagram S1 CONSORT Flow diagram. (PPTX) Click here for additional data file. Checklist S1 CONSORT checklist. (DOCX) Click here for additional data file.

TITLE: Comparison of Transforaminal and Parasagittal Epidural Steroid Injections in Patients With Radicular Low Back Pain ABSTRACT.BACKGROUND:: Epidural steroid injection (ESI), including transforaminal (TF) epidural injections and interlaminar (IL) epidural steroid injections are commonly performed procedures for the management of lumbosacral radicular pain. Parasagittal interlaminar (PIL) approach could enable higher ventral epidural spread, with fewer complications than TF. ABSTRACT.OBJECTIVES:: This study aims to compare the effectiveness of PIL and TF ESI in relieving the pain and disability of patients with lumbosacral pain. ABSTRACT.PATIENTS AND METHODS:: This prospective study enrolled 64 patients, aged between 18 to 75 years, with a diagnosis of low back pain and unilateral lumbosacral radicular pain. The patients were randomized to receive fluoroscopically guided epidural injection, through either the PIL or TF approach. Patients were evaluated for effective pain relief [numerical rating scale (NRS) < 3] by 0 - 10 numeric rating scale (NRS) and functional improvement by the Oswestry Disability Index (ODI). ABSTRACT.RESULTS:: Effective pain relief [numeric rating scale (NRS) < 3] was observed in 77.3% (95% CI: 67‒90.5%) of patients in PIL group and 74.2% (95% CI: 62.4 - 89.4%) of patients in the TF group (P = 0.34), at 4 weeks. Mean NRS score was not significantly different between the PIL group compared to the TF group, at 4 weeks (P = 0.19). Number of patients with improved disability (measured by ODI < 20%) was not significantly different in PIL group (78% of cases) compared to the TF group (76% of cases), at 4 weeks (P = 0.21). There were no adverse effects observed in any of our patients. ABSTRACT.CONCLUSIONS:: The PIL epidural injection is as effective as TF epidural injection in improving pain and functional status, in patients with chronic lumbosacral low back pain, due to disc degeneration. BODY.1. BACKGROUND: Epidural steroid injection (ESI), including transforaminal (TF) epidural injections and interlaminar (IL) epidural steroid injections are commonly performed procedures for the management of lumbosacral radicular pain (1). Although both procedures are minimally invasive and effective (2), there is, however, lack of substantial evidence about the superior efficacy of one of these interventional approaches. The IL epidural injection can be delivered through midline, paramedian, or parasagittal approaches (3). The TF entry can be directed more closely to the assumed site of pathology in the anterolateral (AL) epidural space, requiring less volume of injection than the IL route. Therefore, TF approach is considered more efficacious than the midline IL approach, probably due to better ventral epidural spread (4). In the meantime, the concerns regarding the safety of the TF approach lead to the search for a technically better route, with lesser complications in the IL approach. The parasagittal interlaminar (PIL) route could induce higher ventral epidural spread, with fewer complications than TF (5). Although there is limited evidence in the literature showing the effectiveness of PIL in redirecting epidural injection to ventral space, however, it has not been thoroughly studied compared to the TF approach. BODY.2. OBJECTIVES: This study aims to compare the effectiveness of PIL and TF ESI in relieving the pain and disability of patients with lumbosacral pain. BODY.3. PATIENTS AND METHODS: The study was reviewed and approved by the University Review Board of Shahid Beheshti University of Medical Sciences, Tehran, Iran, and the Ethics Committee of Akhtar Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Information about the trial was given comprehensively, both orally and in written form, to the parents. All patients gave their written informed consents prior to their inclusion in the study, accordingly. BODY.3.1. PATIENT SELECTION: This double blind clinical trial enrolled patients aged between 18 to 75 years, with a diagnosis of low back pain and unilateral lumbosacral radicular pain, due to a contained herniated degenerated disc, with a minimum of 6 months duration, not responding to medications and physical therapies. Magnetic resonance imaging (MRI) was performed to correlate the symptoms and exact disc level protrusion, or contained herniation. Patients were excluded if they had any clinically significant or unstable medical or psychiatric illness, previous surgery on the lumbar spine, facet joint arthropathy, spinal canal stenosis, unstable neurological deficits, or cauda equina syndrome. Those who had received lumbar ESI in the past, were allergic to corticosteroids, taking anticoagulants or had bleeding diathesis, were taking systemic corticosteroids, pregnant women, or those being ESI injected within 30 days of trial, were also excluded. Randomization was performed based on accidental numbers assigned by the computer to one of the groups of the study. Study was blinded to both sides, in which neither the patients, nor the physician evaluating, knew the group of study. Patients were given a code by the computer and the code was only known to the physician, who delivered pocket containing group of patient. Then, the code was registered as anonymous to the physician inside the operation room, who performed one of the procedures, based on the label inside the pocket accompanying the patients. After performing the procedure, the patient was evaluated by another physician, who was also blind to the code and group of patients. BODY.3.2. PARASAGITTAL INTERLAMINAR EPIDURAL STEROID INJECTION PROCEDURE: An 18-gauge, 3.5 inches, Tuohy needle was introduced at the level of disc lesion and advanced in a posterior to anterior direction, vertical to the body surface. After determination of the most lateral place for needle entrance, in fluoroscopy anteroposterior (AP) view, the needle was introduced into the epidural space of the affected side, using the loss-of-resistance and this parasagittal orientation of the needle was maintained throughout the procedure. Bevel direction was positioned toward lateral. BODY.3.3. TRANSFORAMINAL PROCEDURE: In the TF group, a 22-gauge, 3.5 inch Quincke’s needle was introduced at the level of disc lesion, using first an AP and, subsequently, an oblique orientation (15 - 30°) of the fluoroscopy C-arm to achieve the “Scotty Dog” appearance of the lumbar spine and then directed until the needle tip was in the posterior and superior aspect of the intervertebral foramen, as checked in the lateral imaging, and in line with the pedicle on AP view. BODY.3.4. CONFIRMATION OF EPIDURAL STEROID INJECTION: In both groups, once the needle was in position, and after negative aspiration for cerebrospinal fluid and blood, 1 mL contrast dye (OMNIPAQUE ™, GE Healthcare, Hatfield, UK) was injected to confirm the epidural space distribution in the AP view. This was followed by further injection of 3 mL of contrast under fluoroscopy, to confirm the spread of the contrast, as well as to verify that no contrast medium attained the intravascular, subarachnoid, subdural, or intra-discal spread. Lateral images were taken to evaluate the ventral epidural space. Ventral spread was defined as present if contrast travelled along the posterior longitudinal ligament or adjacent to the posterior aspect of the contiguous vertebral body at the level of needle insertion. Perineural spread and segmental spread were also noted on AP view. After epidural space confirmation, 2 mL of triamcinolone (l mL = 40 mg), plus 2 mL of bupivacaine (2 mL = 10 mg) and 6 mL sterile normal saline were injected. All the patients were monitored for at least 30 minutes after procedure. BODY.3.5. DATA RECORDING: Sixty four patients were randomized to receive fluoroscopically guided epidural injection, through either the PIL or TF approach. Patients were evaluated for effective pain relief [numeric rating scale (NRS) < 3] by 0 - 10 NRS and functional improvement by Oswestry Disability Index (6) (ODI < 20%) at 2 and 4 weeks. Any complications or side effects were recorded. BODY.4. RESULTS: In this prospective cohort of 64 patients with chronic low back pain, 32 patients received TF epidural injection and 32 received epidural through PIL. Demographic characteristics are listed in Table 1. There were no significant differences in age, sex, body mass index (BMI), duration of pain, pre-procedure pain score (NRS), and functional status (ODI), prior to procedure between the two groups (P > 0.05) (Table 1). Table 1.BODY. DEMOGRAPHIC CHARACTERISTICS OF THE PATIENTS :a Demographic CharacteristicParasagittal bTF bP Value Age, y 49.2 ± 15.5 50.5 ± 16.6 0.32 Gender (Male/Female) 19/13 20/12 0.52 BMI, kg/m2 22.1 ± 3.4 21.7 ± 3.1 0.17 Duration of pain, mo 8.7 ± 6.5 8.7 ± 7.9 0.67 Pre-procedure NRS 7.1 ± 2.5 7.5 ± 2.4 0.13 Pre-procedure ODI, % 55.2 ± 12.4% 53.6 ± 16.1% 0.082 a Abbreviations: BMI, Body Mass Index; NRS, Numeric Rating Scale; ODI, Oswestry Disability Index. b n = 32. BODY.4.1. EFFECTIVE PAIN RELIEF: Effective pain relief (NRS < 3) was observed in 77.3% (95% CI: 67‒90.5%) of patients in the parasagittal group and 74.2% (95% CI: 62.4‒89.4%) of patients in the TF group (P = 0.34), at 4 weeks (Figure 1). Besides, mean NRS pain score was also compared between the two groups. Mean NRS was not significantly different in the two groups, prior to epidural injection (P > 0.05). Mean NRS score was not significantly different in PIL group, compared to TF, at 4 weeks (P = 0.19) (Figure 1). Figure 1.BODY.COMPARISON OF THE PROPORTION OF PATIENTS ACHIEVING EFFECTIVE PAIN RELIEF IN PARASAGITTAL AND MIDLINE EPIDURAL INJECTION: The NRS < 3 at 4 weeks after PIL or TF epidural injection (left figure); Mean pain score (NRS) during follow-up time between two groups of PIL and TF epidural injection (right figure) and Abbreviations: NRS, numeric rating scale; PIL, parasagittal interlaminar; TF, transformainal. BODY.4.2. FUNCTIONAL IMPROVEMENT DURING FOLLOW UP TIME: Number of patients with improved disability (measured by ODI < 20%) was not significantly different in parasagittal group (78% of cases), compared to the TF group (76% of cases), at 4 weeks (P = 0.21) (Figure 2). Besides, mean ODI score was also compared between the two groups. Mean ODI was not significantly different in the PIL compared to TF group, at 4 weeks follow up (P = 0.15) (Figure 2). There was no adverse effect observed in any of our patients. Figure 2.BODY.COMPARISON OF THE PROPORTION OF PATIENTS IMPROVING DISABILITY INDEX: The ODI was <2 0%, at 2 weeks between the two groups of parasagittal and transforaminal epidural injection (left figure); Mean ODI score during the follow-up time between the two groups of parasagittal and midline epidural injection (right figure). BODY.5. DISCUSSION: Intervertebral disc herniation, spinal stenosis, intervertebral disc degeneration, and failed back syndrome are the most common diagnosis of chronic radicular low back pain. Epidural injections are one of the most commonly performed interventions in managing chronic low back pain. Among various approaches for ESI, TF is considered as target specific and more effective, compared to midline IL ESI (7). This may be due to blind administration of IL or needle placement in the dorsal space, under fluoroscopic guidance, leading to distribution of the drug to the dorsal space, rather than ventral space (8). Botwin et al. (9) evaluated lumbar IL epidural injections in epidurography pattern. They showed that dorsal contrast of flow occurred in 100% of injections; however, ventral spread of the contrast was seen only in 36% of the patients. In another study, Choi and Barbella (10), in an evaluation of contrast patterns of IL epidural injections, showed excellent spread of contrast into the nerve root and the ventral epidural space in all patients, utilizing a paramedian approach. The advantage of TF over midline IL injections is attributed to the enhanced deposition of medication in ventral epidural space, close to the source of pain, with a smaller dose of medication (11). There is evidence suggesting that TF allows for greater ventral epidural spread of corticosteroid (12), and ventral epidural spread of corticosteroid has been associated with higher pain and functional improvements. In our study, there was no significant difference in pain score and functional disability, after 4 weeks of follow up, between PIL and TF epidural injections. This indicates that, in both approaches, the drug is able to reach the ventral space. The existing data suggests long-term efficacy benefits are greater for TF, compared to IL (13-15). However, it remains unclear if TF ESI result in clinically or statistically significant improvements in pain and functional outcomes, compared to IL (16). Although we did not observe any complications of TF or PIL ESI, however, other literature reviews have indicated that TF are more often implicated in complications, compared to IL ESI, including intravascular injection in up to 23% of lumbar epidural injection cases (17), which can lead to spinal cord infarction and paralysis. A meta-analysis (18) showed that TF resulted in better short-term pain improvement and fewer long-term surgical interventions than midline IL ESI. However, TF injection complications risk must be taken into consideration (19). There have been reports of pneumocephalus during TF. The complication of dural puncture is documented in the context of a lumbar TF (20). The incidence of vascular penetration, during contrast confirmed fluoroscopically guided TF epidural injections have been reported to 8.9‒21.3%, depending on the level of injection (21). Previous study demonstrates a high incidence of intravascular injections in TF lumbosacral epidural injections (22). Even in severe cases, studies have presented a case of quadriparesis and brainstem herniation after selective cervical TF (23). The TF, compared to IL ESI, are associated with a 12-fold increased risk of intradiscal injection (24), which can potentially weaken the disc or lead to discitis (25). Other methods, such as intradiscal ozone injection, have been shown to have a positive effect on disk herniation (26). In conclusion, PIL epidural injection is as effective as TF epidural injection in improving pain and functional status in patients with chronic lumbosacral low back pain, due to disc degeneration. Parasagittal approach holds the advantage of avoiding the risk of complications associated with the TF approach.

TITLE: Liraglutide suppresses non-esterified free fatty acids and soluble vascular cell adhesion molecule-1 compared with metformin in patients with recent-onset type 2 diabetes ABSTRACT.BACKGROUND: It has been suggested that liraglutide could have an impact on glucose and lipid metabolism disorder and adhesion molecule activation, which may play important roles in the vascular damage of diabetes. In this study, we examined the effects of liraglutide versus metformin on non-esterified free fatty acids, beta-cell insulin secretion, and adhesion molecule levels in patients with recent-onset type 2 diabetes mellitus. ABSTRACT.METHODS: In this study, 60 patients newly diagnosed with type 2 diabetes mellitus (mean age 33.97 ± 5.67 years) were randomly assigned to receive once-daily subcutaneous liraglutide or oral metformin. Before the study and after the 8-week treatment period, a 75 g oral glucose tolerance test was performed. Plasma glucose, lipids and lipoprotein, plasma insulin, glycaemic and insulin responses, non-esterified free fatty acids (NEFA), and soluble vascular cell adhesion molecule-1 (sVCAM-1) levels were evaluated. ABSTRACT.RESULTS: After 8 weeks, 120 min of NEFA (155 ± 125 vs 99 ± 73 µmol/L, P = 0.026) and the levels of sVCAM-1 (465 ± 136 vs 382 ± 131 ng/ml, P = 0.013) significantly decreased, while the early phase insulin secretion index (24.94 [7.78, 38.89] vs. 31.13 [17.67, 59.09], P = 0.031), fasting plasma insulin (104 [51, 123] vs 113 [54, 171] mIU/L, P = 0.015), 60 min plasma insulin (326 [165, 441] vs 471 [334, 717] mIU/L, P = 0.005), 120 min plasma insulin (401 [193, 560] vs 500 [367, 960] mIU/L, P = 0.047), and insulin area under the curve (AUCins) (648 [321, 742] vs 738 [451, 1118] mIU/L, P = 0.005) remarkably increased for patients in the liraglutide treatment group. The levels of sVCAM-1 dramatically decreased after 8 weeks of liraglutide treatment (503 ± 182 vs 382 ± 131 ng/ml, P = 0.046) compared to that of the metformin treatment group. At the same time, the differences before and after liraglutide treatment in 120 min of NEFA (− 32 [− 96, − 5] vs 5 [− 35, 38] µmol/L, P = 0.033) and AUCins (738 [451, 1118] vs 594 [357, 1216] mIU/L, P = 0.014) were remarkably enhanced compared to that of the metformin therapy. Nevertheless, there were no significant differences in fasting NEFA after liraglutide or metformin treatment. The reduction of 120 min NEFA (ΔNEFA) was positively correlated with the decrease of sVCAM-1 (ΔsVCAM-1) after 8 weeks of liraglutide treatment (r = 0.523, P = 0.003). ABSTRACT.CONCLUSIONS: Our results demonstrate that liraglutide administration is more effective than metformin in reducing 120 min NEFA and suppressing sVCAM-1 levels for recent-onset type 2 diabetes mellitus. We suggest that this outcome may be because liraglutide is associated with potentiating insulin secretion capacity, inhibiting vascular inflammatory cytokines, and antagonizing atherosclerosis. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12933-018-0701-4) contains supplementary material, which is available to authorized users. BODY.INTRODUCTION: It has been well documented that atherosclerosis is the major cause of death in type 2 diabetic patients [1, 2]. Dyslipidaemia plays an important role in the pathogenesis of atherosclerosis and even to insulin resistance [3, 4]. Dyslipidaemia is associated with increased lipolysis and the release of higher amounts non-esterified fatty acids (NEFAs) into the bloodstream [5]. Hyperglycaemia promotes lipolysis and leads to chronic exposure to NEFA [6]. Several studies have shown that the elevated serum level of NEFA contributes to vascular damage in diabetes [7, 8]. Plasma NEFAs promote a systemic insulin resistance state and are correlated with inflammation in atheromatous plaques [5, 7]. The levels of NEFAs seem to be modified by dietary or therapeutic intervention. Glucagon-like peptide-1 (GLP-1) has recently received attention as a novel drug in the antidiabetic field [9]. The long-acting GLP-1 analogue liraglutide (LRG) exhibits pleiotropic effects on glucolipid metabolism, β-cell insulin secretion and anti-atherosclerosis [10–15]. Nevertheless, the precise mechanism by which liraglutide modulates lipids metabolism remains unclear. Therefore, the aim of this study is to evaluate whether liraglutide could be more effective at suppressing lipolysis, ameliorating glucose and lipid metabolism, enhancing beta-cell functions, and inhibiting vascular inflammatory markers compared to metformin in recent-onset type 2 diabetes mellitus. BODY.SUBJECTS AND METHODS: BODY.ETHICAL APPROVAL: Ethical approval was granted by the Institutional Review Board of Xiamen University Affiliated Zhongshan Hospital according to Helsinki Declaration II. Written informed consent was signed by and obtained from each participant. BODY.PARTICIPANTS: Participants were recruited between October 2015 and December 2016 at Zhongshan Hospital Xiamen University in P. R. China. The inclusion criteria were set to select participants: the patients had an initial diagnosis of type 2 diabetes mellitus who met the World Health Organization criteria for type 2 diabetes and were aged 18–40 years, with a body mass index (BMI) of 25–35 kg/m2, HbA1c of 6.5–9%, and fasting serum triglycerides of 0.5–5 mmol/L. Main exclusion criteria included the following: type 1 diabetes, recent acute complications including diabetic ketoacidosis and hyperglycaemic hyperosmolar state, impaired liver function, impaired renal function (creatinine clearance < 60 ml/min) [16], pregnancy and breast bleeding, and other factors that affect glucose and lipid changes. BODY.STUDY DESIGN AND PROCEDURES: This study is a randomized, parallel, active comparator trial in 60 participants. The treatment order of liraglutide or metformin was determined through random assignment (1:1). All individuals who received liraglutide treatment started injection doses at 0.6 mg/day. After 1 week of injection, the dose was increased to 1.2 mg/day. Participants in another group who received metformin received an oral dose in the range of 1–2 g/day. After an overnight fast, a 75 g oral glucose tolerance test (OGTT) was conducted for each participant. Blood samples were drawn before and 30, 60, 120 min after OGTT, respectively. Participants in the liraglutide treatment group were instructed to administer the Flexpen device. Participants were also informed of medication precautions before metformin treatment. All participants received lifestyle intervention health education from professional nurse. The total planned treatment period was 8 weeks. There were a total of 3 follow-up visits, once per month. The plasma glucose, blood pressure, body weight, waist circumference and hip circumference were measured, and adverse events were monitored by investigators. At the end of the trial, the clinical and laboratory indices were assessed, as previously described. BODY.CLINICAL MEASUREMENT AND LABORATORY TEST: Weight, waist circumference (WC), and hip circumference were measured by professional nurses. Participants removed clothing and shoes prior to measurements. WC was measured midway between the lowest rib and the top of the iliac crest. Hip circumference was measured around the peak of the buttocks. Body mass index (BMI) was calculated as the body weight (BW) in kilograms divided by the square of the patient’s height in metres. Measurements of systolic blood pressure and diastolic blood pressure (SBP and DBP) were performed 2 times with a mercury manometer on the right arm after 5 min in the sitting position. The mean values of measurements were recorded. Participants were instructed to refrain from excessive physical exercise and to eat normally 3 days before the test, with no less than 200 grams of carbohydrates daily. After fasting overnight (8–10 h), a 2-h OGTT test was performed on each subject. Fasting plasma glucose (FPG), 30 min PG, 60 min PG, and 120 min PG were measured by the hexokinase method. Subjects’ renal and liver functions, plasma lipids and lipoprotein concentrations including triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were assayed using standard methods (Roche cobas8000 automatic biochemical analyser). HbA1c was analysed by HPLC (Bio-Rad, Inc., Hercules, CA, USA). Plasma insulin levels were measured using the electrochemiluminescence immunoassay (ECLI). NEFA levels were measured in the Xiamen Diabetes Hospital using Abbott c16000 automatic biochemical analyser. Soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) were measured in the plasma of patients using quantikine ELISA kits (R&D systems, Inc., Minneapolis, USA). Plasminogen activator inhibitor 1 (PAI-1) concentrations were assessed using ELISA assays (Multi sciences, Inc., Hangzhou, PRC). The intra-assay and inter-assay coefficients of variation (CV) of the ELISA kits mentioned above were all less than 10%. The early phase insulin secretion index was calculated as (ΔI30/ΔG30) = ([insulin at 30 min] − [insulin at 0 min])/([glucose at 30 min] − [glucose at 0 min]) [17]. The area under the curve (AUC) for plasma insulin during the OGTT was calculated using the trapezoidal rule [18]. Deltas (Δ) are presented as the difference before and after treatment, which were available for the variables ΔNEFA, ΔsVCAM-1, ΔAUCins, and ΔI30/ΔG30. Deltas were also considered to express the difference between 120 min values and fasting values in ΔTC and ΔLDL-C. BODY.STATISTICAL ANALYSIS: SPSS version 21 (SPSS software, IBM Inc., USA) and GraphPad Prism version 5.0 (GraphPad software, Inc., La Jolla, CA, USA) were utilized for statistical analysis and the construction of graphs. The normal distribution data are presented as the mean ± standard deviation. Comparisons of basic characteristics between groups were made using an unpaired t test. Comparisons of baseline and post-treatment data in the same group were made using a paired t test. The skewed distribution variables are expressed as the median (interquartile rang), and the Mann–Whitney U test or Wilcoxon signed rank test was performed. The Mann–Whitney U test was used to determine the differences from baseline with a final measurement at 8 weeks for 120 min NEFA, insulin secretion capacity including fasting insulin, 30 min insulin, 60 min insulin, 120 min insulin, ΔI30/ΔG30, and AUCins between the liraglutide and metformin group. At the same time, the Mann–Whitney U test was carried out to determine the differences between 120 min values and fasting values on TC and LDL-C in the metformin group. Spearman rank correlations were performed to determine the relationship of ΔNEFA with ΔsVCAM-1, ΔAUCins and ΔI30/ΔG30 after 8 weeks of treatment. The Chi squared (χ2) test was used for categorical variables. Two-tailed significance was set at P < 0.05. BODY.RESULTS: BODY.COMPARISONS OF CLINICAL AND LABORATORY CHARACTERISTICS OF THE STUDY PARTICIPANTS: The patient characteristics grouped by either liraglutide or metformin treatment are presented in Table 1. In the baseline analysis, the differences in participants’ age, BW, BMI, SBP, DBP, WC and hip circumference between the two groups were not statistically significant. HbA1c, FPG, 60 min PG, 120 min PG, NEFA, lipids and lipoprotein concentrations (including TC, TG, LDL-C, HDL-C), insulin secretion capacity, sVCAM-1, sICAM-1, and PAI-1 were similar between the two groups, but 30 min PG was higher at baseline in the liraglutide treatment group.Table 1Baseline characteristics of the study participants VariableLiraglutide (n = 30)Metformin (n = 30)P-valueAge (years) 32.67 ± 5.46 35.27 ± 5.85 0.219 Male/female (n) 20/10 20/10 1.000 BW (kg) 81 ± 17 72 ± 11 0.104 BMI (kg/m2) 28.63 ± 3.86 26.16 ± 3.1 0.063 Waist circumference (cm) 92.20 ± 11.99 88.27 ± 8.22 0.304 Hip circumference (cm) 99.87 ± 6.02 96.33 ± 5.77 0.112 SBP (mmHg) 122 ± 16 120 ± 11 0.643 DBP (mmHg) 79 ± 12 81 ± 7 0.719 Cholesterol (mmol/L)  Fasting value 4.79 ± 0.87 5.02 ± 0.79 0.444  120 min value 4.45 ± 1.02 4.55 ± 0.81 0.777 Triglycerides (mmol/L)  Fasting value 1.82 ± 0.74 1.99 ± 0.74 0.539  120 min value 2.07 ± 1.23 1.97 ± 0.68 0.802 LDL-C (mmol/L)  Fasting value 3.61 ± 0.82 3.78 ± 0.68 0.535  120 min value 3.16 ± 0.70 3.28 ± 0.70 0.639 HDL-C (mmol/L)  Fasting value 1.12 ± 0.29 1.20 ± 0.26 0.448  120 min value 1.04 ± 0.35 1.07 ± 0.23 0.793  HbA1c (%) 8.49 ± 1.81 7.77 ± 1.44 0.238  FPG (mmol/L) 9.40 ± 2.32 8.45 ± 1.57 0.198  30 min PG (mmol/L) 15.43 ± 2.96 13.03 ± 2.72 0.028  60 min PG (mmol/L) 18.19 ± 3.60 16.43 ± 4.13 0.224  120 min PG (mmol/L) 17.68 ± 4.38 14.47 ± 5.05 0.074  Fasting insulin (mIU/L) 104 (51, 123) 76 (60, 150) 0.724  30 min insulin (mIU/L) 271 (136, 298) 211 (118, 444) 0.917  60 min insulin (mIU/L) 326 (165, 441) 368 (242, 731) 0.152  120 min insulin (mIU/L) 401 (193, 560) 475 (286, 1060) 0.178  AUCins (mIU/L) 648 (321, 742) 615 (381, 1167) 0.520  ΔI30/ΔG30 24.94 (7.78, 38.89) 30.18 (10.4, 53.75) 0.272 NEFA (μmol/L)  Fasting value 620 ± 468 485 ± 225 0.325  120 min value 155 ± 125 101 ± 53 0.309  sVCAM-1 (ng/ml) 465 ± 136 485 ± 122 0.669  sICAM-1 (ng/ml) 113 ± 40 168 ± 110 0.085  PAI-1 (ng/ml) 69.52  ± 45.75 51.34  ± 38.87 0.227 Data are expressed as mean ± standard deviation or median (interquartile rang) BW body weight, BMI body mass index, SBP systolic blood pressure, DBP diatolic blood pressure, LDL-C low-density lipoprotein cholesterol, HDL-C high-density lipoprotein cholesterol, HbA1c glycated haemoglobin, FPG fasting plasma glucose, AUCins insulin area under the curve, NEFA non-esterified fatty acids, sVCAM-1 soluble vascular cell adhesion molecule-1, sICAM-1 soluble intercellular adhesion molecule-1, PAI-1 plasminogen activator inhibitors-1 After 8-week metformin treatment, fasting total cholesterol (TC) and low density lipoprotein cholesterin (LDL-C) were remarkably lower compared to baseline (5.02 ± 0.79 vs 4.45 ± 0.28 mmol/L, P = 0.006), (3.78 ± 0.68 vs 3.18 ± 0.88 mmol/L, P = 0.012). At the same time, metformin therapy also reduced 120 min TC (4.55 ± 0.81 vs 4.10 ± 1.21 mmol/L, P = 0.036) and 120 min LDL-C (3.28 ± 0.7 vs 2.76 ± 0.91 mmol/L, P = 0.008) after OGTT. However, in the present study, there were no significant differences between 120 min values and fasting values in ΔTC and ΔLDL-C with respect to metformin treatment. Furthermore, no significant differences were found in plasma lipids and lipoprotein after liraglutide treatment compared with the basal values (shown in Additional file 1: Table S1). We found a significant reduction in waist circumference (92 ± 12 vs 88 ± 11 cm, P = 0.001) and body weight (81 ± 17 vs 78 ± 16 kg, P = 0.001), as well as BMI (28.63 ± 3.86 vs 27.67 ± 3.62 kg/m2, P = 0.001), with liraglutide treatment. Metformin treatment also showed a slight downtrend in BW and BMI, although the difference was not significant (shown in Additional file 1: Table S2). BODY.LIRAGLUTIDE TREATMENT ELEVATED INSULIN SECRETION CAPACITY AND DECREASED PLASMA GLUCOSE: Early phase insulin secretion index (ΔI30/ΔG30) (24.94 [7.78, 38.89] vs 31.13 [17.67, 59.09], P = 0.031), fast plasma insulin (104 [51, 123] vs 113 [54, 171] mIU/L, P = 0.015), 60 min plasma insulin (326 [165, 441] vs 471 [334, 717] mIU/L, P = 0.005), 120 min plasma insulin (401 [193, 560] vs 500 [367, 960] mIU/L, P = 0.047), and insulin area under the curve (AUCins) (648 [321, 742] vs 738 [451, 1118] mIU/L, P = 0.005) significantly increased with liraglutide treatment. The differences in AUCins (ΔAUCins) before and after liraglutide treatment (738 [451, 1118] vs 594 [357, 1216] mIU/L, P = 0.014) were remarkably enhanced compared to that in metformin therapy, but differences in ΔI30/ΔG30 before and after liraglutide treatment (6.01 [1.79, 28.46] vs − 2.62 [− 14.85, 8.76], P = 0.065) were not significant between the two groups (Table 2). In fact, both liraglutide (8.49 ± 1.81 vs 6.92 ± 0.81%, P = 0.002) and metformin treatment (7.77 ± 1.44 vs 6.43 ± 0.71%, P = 0.001) could reduce HbA1c values. FPG, 30 min PG, 60 min PG and 120 min PG all decreased after liraglutide treatment, but only FPG was significantly reduced after metformin treatment (shown in Additional file 1: Table S2).Table 2Comparisons of sVCAM-1, 120 min NEFA, AUCins and ΔI30/ΔG30 levels after 8-week treatment between liraglutide and metformin group VariableLiraglutideMetforminDifferenceP-valuesVCAM-1 (ng/ml) 382 ± 131 503 ± 182 0.046 120 min NEFA (μmol/L)a − 32 (− 96, − 5) 5 (− 35, 38) − 20.5 (− 46.5, 31.5) 0.033 AUCins (mIU/L)a 738 (451, 1118) 594 (357, 1216) 39 (− 33, 227) 0.014 Δa 6.01 (1.79, 28.46) − 2.62 (− 14.85,8.76) 4.56 (− 4.82, 12.99) 0.065 Data are expressed as mean ± standard deviation or median (interquartile rang) sVCAM-1 soluble vascular cell adhesion molecule-1, NEFA non-esterified fatty acids, AUCins insulin area under the curve ΔI30/ΔG30 = [(insulin at 30 min) − (insulin at 0 min)]/[(glucose at 30 min) − (glucose at 0 min)] aDifference between pre-treatment and post-treatment BODY.LIRAGLUTIDE TREATMENT INHIBITED 120 MIN NON-ESTERIFIED FREE FATTY ACIDS: After 8 weeks, there was a progressive decrease in the levels of 120 min NEFA (155 ± 125 vs 99 ± 73 µmol/L, P = 0.026) after liraglutide treatment. Compared with metformin therapy, the reduction in 120 min NEFA (ΔNEFA) remarkably increased after 8-week liraglutide treatment [− 32 (− 96, − 5) vs 5 (− 35, 38) µmol/L, P = 0.033] (Table 2). The changes in 120 min NEFA showed a slight increase after metformin therapy (Fig. 1). There were no significant changes in fasting non-esterified free fatty acids after liraglutide and metformin treatment.Fig. 1a 120 min non-esterified fatty acid (NEFA) before and after 8-week treatment with liraglutide and metformin. b The difference of NEFA (∆ NEFA) between baseline and after 8-week treatment with liraglutide and metformin *P <0.05 vs the metformin group BODY.LIRAGLUTIDE TREATMENT INHIBITED VASCULAR INFLAMMATORY MARKER LEVELS: We found a significant reduction in sVCAM-1 (465 ± 136 vs 382 ± 131 ng/ml, P = 0.013) after liraglutide treatment compared with baseline. Compared to metformin therapy, the levels of sVCAM-1 were remarkably suppressed after 8-week liraglutide treatment (382 ± 131 vs 503 ± 182 ng/ml, P = 0.046) (Fig. 2, Table 2). However, there were no significant differences in sICAM-1 and PAI-1 levels before and after liraglutide and metformin therapy (shown in Additional file 1: Table S3). In all subjects combined, the reduction of 120 min NEFA (ΔNEFA) was positively correlated with the decrease of sVCAM-1 (ΔsVCAM-1) after 8-week liraglutide treatment (r = 0.523, P = 0.003), which was not significantly negatively correlated with the increase of AUCins (ΔAUCins) (r = − 0.286, P = 0.125) and ΔI30/ΔG30 (r = − 0.150, P = 0.429) (shown in Additional file 1: Table S4).Fig. 2Changes of soluble vascular cell adhesion molecule-1 (sVCAM-1) before and after 8-week treatment with liraglutide and metformin BODY.DISCUSSION: Our results indicate that prolonged liraglutide treatment for 8 weeks effectively inhibited 120 min non-esterified free fatty acids (NEFAs). We utilized a 75 g oral glucose tolerance test (OGTT) to evaluate the changes of NEFA, which should avoid the effects of a high-fat diet on postprandial lipid metabolism. As observed in this study, NEFA levels gradually decreased after glucose uptake, and 120 min NEFAs are dramatically lower than fasting NEFAs. Furthermore, our data show that liraglutide exhibits greater stimulation on the insulin secretion capacity, including early phase insulin secretion, fast plasma insulin, 60 min plasma insulin, 120 min plasma insulin and insulin area under the curve (AUCins) relative to those at baseline. Previous research has shown that insulin could suppress chylomicron synthesis from human jejunal explants [19]. Additionally, circulating FFAs derived from chylomicron are reduced in the hyperinsulinaemia state, as insulin prevents lipolysis in the regulation of hepatic and intestinal lipoprotein production [20, 21]. Our data demonstrated that after the 8-week treatment period, the reduction in 120 min NEFA (ΔNEFA) was greater increased, and the increase in AUCins (ΔAUCins) was also enhanced in liraglutide treatment relative to metformin treatment. Consequently, in this study, liraglutide therapy exhibited beneficial effects in ameliorating the β-cell secretion capacity and suppressing free fatty acid production. However, the precise mechanisms by which glucagon-like peptide-1 affects lipids metabolism are less certain. It has been hypothesized that enterocyte GLP-1 receptor signalling is essential for postprandial lipoprotein synthesis and secretion, reduces intestinal lipid production and absorption, and prevents the postprandial increases in triacylglycerol, cholesterol and apo-B48 levels [22]. Moreover, GLP-1 is related to the direct modulation of fatty acid binding protein 2 (FABP2), which is required for the formation of apo-B48 containing chylomicrons [23]. In addition, even through enhanced insulin secretion, GLP-1 induced prolonged reductions in NEFA concentrations after meals, suppressed postprandial rises in ApoCIII [24]. ApoCIII is a small glycoprotein synthesized in the liver and intestine that resides predominantly on the surface of ApoB-containing lipoproteins and HDL. ApoCIII inhibits lipoprotein lipase activity and interferes with the receptor-mediated uptake of TG-rich lipoproteins, therefore delaying the clearance of TG-rich lipoproteins [25, 26]. On the other hand, a long-term increase in NEFA levels has been associated with ROS accumulation, which causes mitochondrial stress and leads to β-cell apoptosis [27, 28]. In a catch-up growth rat model by re-feeding with high-fat diet, liraglutide prevented the increase of plasma NEFA, increased insulin secretion, increased islet pancreatic duodenal homeobox-1 (Pdx-1) and B cell lymphoma-2 (Bcl-2) expression, and reduced procaspase-3 transcription and Caspase-3 p11 subunit protein expression, which suggested that liraglutide treatment could antagonize β-cell apoptosis caused by elevated NEFA [11]. Dyslipidaemia has been reported to affect vascular endothelial function by the inflammatory pathway [29]. Raised non-esterified fatty acids impair insulin’s effect on glucose uptake in skeletal muscle and could thus have detrimental effects on the vascular endothelium, which leads to premature cardiovascular disease [30]. As reported previously, the adhesion molecules usually act as biomarkers, which mediate interactions between leukocytes and the vascular endothelium, shed from cell surfaces and become soluble isoforms in the blood, namely, sVCAM-1 and sICAM-1 [31]. Our study has shown that the levels of the vascular inflammatory marker sVCAM-1 are dramatically suppressed after liraglutide treatment compared to metformin treatment. However, we did not identify appreciable differences in the levels of sICAM-1 and PAI-1 after liraglutide therapy. Considerable evidence has demonstrated that sVCAM-1 plays an important role in the pathophysiological mechanisms of atherosclerosis [32]. During a 10-year follow-up, a longitudinal study revealed that elevated circulating sVCAM-1 concentrations in females with a history of gestational diabetes mellitus (GDM) not only represented the earliest high risk stage for developing type 2 diabetes, but also reflected the early stage of the pathway for the manifestation of future cardiometabolic disorders [33]. It is widely known that atherosclerosis leads to macroangiopathy and is responsible for most deaths in patients with diabetes [34]. Liraglutide was shown to significantly reduce the risk of major cardiovascular events in the large prospective LEADER trial [12, 35–37]. However, the precise mechanisms behind the antiatherogenic effect of liraglutide are not entirely clear, although several hypotheses have been proposed. First, it has been shown that in cultured human aortic endothelial cells (HAECs), liraglutide reduces the protein expression of VCAM-1 in response to TNFα and LPS stimulation and increases intracellular calcium and cAMP levels, leading to the phosphorylation of AMPK, which is an evolutionarily conserved fuel and stress-sensing enzyme that can be activated by calmodulin dependent protein kinase kinase-b (CAMKKb), and then downregulates monocyte adhesion, which presumably inhibits atherogenesis [38, 39]. Second, in a cellular model of human umbilical vein endothelial cells (HUVECs) obtained from umbilical cords of women affected by GDM, liraglutide exposure significantly mitigated TNFα induced endothelial monocyte adhesion as well as VCAM-1 and ICAM-1 expression, reduced the phosphorylation of the MAPK42/44 signalling pathway, inhibited NF-kB p65 nuclear translocation, and decreased peroxynitrite levels and endothelial microvesicle (EMV) release [40]. Third, after 8-week metformin treatment, TC and LDL-C were remarkably lower compared to baseline, but no significant differences were found between 120 min values and fasting values in ΔTC and ΔLDL-C. Moreover, no significant differences were found in plasma TC and LDL-C after liraglutide treatment. We presumed that liraglutide exert antiatherogenic mechanism, which might be not related to a decrease of LDL-C levels but might be related to an enhancement of HDL anti-inflammatory capacity [41]. We carried out a Spearman rank correlation assay and the data showed that the reduction of 120 min NEFA (ΔNEFA) was positively correlated with the decrease of sVCAM-1 (ΔsVCAM-1) after 8-week liraglutide treatment. Our results suggest that liraglutide has beneficial effects on suppressing plasma NEFA as well as sVCAM-1 levels, which is likely to indicate that liraglutide could protect the endothelia by inhibiting monocyte cell adhesion and sVCAM-1 activation and reducing lipocyte oxidative stress and free fatty acid production, revealing an antiatherogenic effects [42, 43]. The strengths of this study include the randomized, parallel, active comparator design and the similarities between the liraglutide and metformin groups at baseline. To the best of our knowledge, this is the first study to verify that liraglutide treatment is more effective than metformin treatment for recent-onset type 2 diabetes mellitus in reducing NEFA after glucose intake and suppressing sVCAM-1 levels at the same time. However, this study has certain limitations. First, it has a non-blinded design, lacks a non-treatment control group, and has a small sample size for the study. Second, further studies are needed to reveal the relevant signalling pathways by which liraglutide might influence glucose and lipid metabolism, regulate vascular endothelial function, and antagonize atherosclerosis. BODY.CONCLUSIONS: In brief, this study demonstrated that liraglutide administration is more effective in diminishing 120 min NEFAs and suppressing sVCAM-1 levels than metformin in young patients with recent-onset type 2 diabetes mellitus. This outcome could be associated with potentiating insulin secretion capacity and antagonizing vascular inflammatory cytokines. Therefore, these results suggest that liraglutide may exert a protective effect on alleviating vascular damage, antagonizing atherosclerosis, and reducing the risk of cardiovascular disease in the course of type 2 diabetes. BODY.ADDITIONAL FILE: Additional file 1: Table S1. Changes of plasma lipids and lipoprotein metabolism parameters before and after 8-week treatment with liraglutide and metfotmin. Table S2. Comparisons of body weight, plasma glucose and insulin secretion capacity before and after 8-week treatment between two groups. Table S3. Comparisons of sVCAM-1,sICAM-1,PAI-1 levels before and after 8-week treatment between two groups. Table S4. Spearman rank correlations in ΔNEFA with ΔsVCAM-1, ΔAUCins and ΔI30/ΔG30 after 8-week treatment.

TITLE: Evaluation of Rhubarb Supplementation in Stages 3 and 4 of Chronic Kidney Disease: A Randomized Clinical Trial Objective. To evaluate the efficacy and safety of Rhubarb supplementation in patients of chronic kidney disease. Material and Methods. This study was a prospective comparative study conducted in patients of chronic kidney disease (stages 3 & 4) attending Renal Clinic of Department of Medicine, JN Medical College & Hospital, AMU, Aligarh. Patients were randomly divided into two interventional groups. Group I (Control) was given conservative management while Group II (Rhubarb) received conservative management along with Rhubarb capsule (350 mg, thrice daily) for 12 weeks. Haemogram and renal function tests were measured at 0, 4, 8, and 12 weeks of treatment. Results. There was progressive improvement in clinical features in both the groups after 12 weeks of treatment but Rhubarb group showed more marked improvement as compared to control group. Both groups showed gradual improvement in the biochemical parameters as compared to their pretreated values which was more marked in Rhubarb supplemented group. There was reduction in blood glucose, blood urea, serum creatinine, and 24 hour total urine protein (TUP). There was increase in haemoglobin, 24 hour total urine volume (TUV), and glomerular filtration rate (GFR). There was no statistical difference in two groups with respect to side effects (P > 0.05). Conclusion. Rhubarb supplementation improved the therapeutic effect of conservative management in stage 3 and stage 4 patients of chronic kidney disease. BODY.1. INTRODUCTION: According to the National Kidney Foundation's Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines [1], chronic kidney disease is defined as kidney damage or glomerular filtration rate (GFR) <60 mL/min/1.73 m2 for 3 months or more, irrespective of cause. The prevalence of CKD in SEEK-India cohort was approximately 17.2% with ~6% having CKD stage 3 or worse [2]. Low protein diet-LPD (0.6 g/kg BW/day) as well as very low protein diet-VLPD (0.3 g/kg BW/day) decreases the accumulation of nitrogen waste products while maintaining an adequate nutritional status [3, 4]. The ideal treatment for CKD-ESRD (end stage renal disease) is renal replacement therapy (RRT) which includes renal transplantation and maintenance dialysis. Since these modalities are costly, required lifelong, not suitable for many patients, associated with many complications, and out of reach of 95–99% of patients, they are managed on conservative therapy [5]. Rhubarb belongs to genus Rheum in the family Polygonaceae. Important derivatives from Rhubarb are anthraquinones like rhein, emodin, and aloe emodin [6]. In CKD, these help in the elimination of nitrogenous products through the alimentary canal and regulation of water and electrolytes metabolism [7]. The abnormal expression of aquaporins (AQPs) could lead to less absorption of water in colon or more secretion of intestinal juice, suggesting that AQPs might be one kind of the effector molecules [8]. Chrysophanol and emodin inhibit the genetic transcription and translation of AQP2 gene. Rhubarb anthraquinones have the ability to downregulate AQP4 expression also [9]. In addition, effect of rhubarb was highly associated with the increasing serotonin levels and serotonin receptors in duodenum [10]. Rhein inhibits the transforming growth factor-beta 1 (TGF-β1) and fibronectin expression in renal tissue, thereby inhibiting extracellular matrix (ECM) deposition [11]. Emodin decreased the gluconeogenesis of renal tubular cells and diminished the ATP content of epithelial mitochondria. Both the Na+/K+-ATPase and Ca2+-ATPase activities of the epithelial cell were attenuated during the administration of emodin in an in vitro study [12]. In a prospective clinical trial conducted in 151 patients with chronic renal failure, the progression rate of renal failure was slowed in patients treated with rhubarb. There was increase in both the plasma albumin and transferrin level, pointing towards an improved nutritional status [7]. The aim of our study was to evaluate the efficacy and safety of rhubarb supplementation in patients of chronic kidney disease. BODY.2. MATERIAL AND METHODS: BODY.2.1. PATIENTS: The present study was conducted from June 2012 to September 2013 in patients of chronic kidney disease attending Renal Clinic of a tertiary care centre of north India. It was a randomized, prospective, double blinded, and parallel group study. The approval for the study was taken from Institutional Ethics Committee. The study is registered under Clinical Trial Registry of India with registration number CTRI/2012/09/002947 (registered on 03/09/2012). Written and informed consent was taken from all patients before enrolling in the study. The diagnosis of CKD was made on the basis of detailed clinical history, physical examination, and investigations. BODY.2.2. INCLUSION CRITERIA: Patients having CKD (stages 3-4), of age 20–60 years, and of either sex were included in the study. BODY.2.3. EXCLUSION CRITERIA: Patients of end stage renal disease (ESRD), on dialysis, pregnant, terminally ill, immunocompromised, or of severe renal pathology such as malignancy were excluded from the study. BODY.2.4. SAMPLE SIZE (:n) n = (z 2/e 2)pq, where z = level of confidence interval at 95%, so z = 1.96; e = acceptable error; p = prevalence (prevalence assumed as 17.2% according to SEEK-India cohort study) [2]; and q = 1 − p. Hence, sample size (n) = [(1.96∗1.96)/(0.09∗0.09)]∗[0.172∗0.828] = 67.54. So, sample size of 68 is minimum required for each group. Taking into consideration a 15% dropout rate, 80 patients were recruited in each group. The power of the study using the study results (GFR) is 100%. BODY.2.5. STUDY DESIGN: Out of 180 assessed patients, 160 patients were enrolled in the study. Fifteen patients (9 of Group I and 7 of Group II) failed to report on subsequent visits and were excluded from the study. Enrolled patients were randomized into two groups at a ratio of 1 : 1 using table generated by random allocation software. The randomization table had 20 subjects in each block to minimize the disparity between the two groups with respect to number of patients at any time of study. After final diagnosis, applying inclusion and exclusion criteria, patients were included in the study. Group I (Control) patients received conservative management of CKD along with placebo while Group II (Rhubarb) patients received conservative management of CKD along with Rhubarb capsule (350 mg) thrice daily (Figure 1). Both groups received treatment for 12 weeks. In conservative management treatment given was renal diet and telmisartan (40 mg OD). All the enrolled patients were regularly followed with haemogram and renal function tests at 0, 4, 8, and 12 weeks of treatment. The primary outcome in this study, that is, improvement in renal functions was assessed by blood urea, serum creatinine, 24 hour total urine protein (TUP), 24 hour total urine volume (TUV), and GFR while secondary outcomes were haemoglobin percent, fasting blood glucose, postprandial blood glucose, serum potassium, and serum calcium. BODY.2.6. SAFETY ASSESSMENTS: All adverse events experienced by a patient or observed by the investigator were recorded on standard ADR reporting forms of CDSCO at each visit. Rhubarb is reported to have laxative effect [7]. Adverse drug reaction's causality assessment was done using Naranjo's Scale [13] and severity assessment by Modified Hartwig & Siegel Scale [14]. A physical examination, including vital signs, was performed at the start of study and at each visit. Additional routine laboratory safety tests like liver function tests (LFT), ECG, and Chest X ray were performed wherever required. BODY.2.7. STATISTICAL ANALYSIS: The values were expressed as mean ± SD. Statistical significance between pre- and posttreatment values in each group was calculated using Student's Paired t-test. Statistical significance between groups was calculated using unpaired t-test. P < 0.05 was considered significant. Statistical analysis was done using SPSS-20 software. The effect size calculated using improvement in serum creatinine was 0.2. BODY.3. RESULT: 71 (41 M, 30 F) patients mean aged 45 years (range 22–58 years) were of Group I and 73 (42 M, 31 F) patients mean aged 45 years (range 21–59 years) were of Group II. The distribution of patients was almost similar and no significant difference (P > 0.05) was seen between the groups. None of the patients in either group required dialysis and there was no mortality in either group. As per GFR (mL/min per 1.73 m2), patients belonged to stage 3 (19 and 20 in Group I and II resp.) and stage 4 (52 and 53 in Group I and II resp.) CKD in both the groups. The causes of CKD in groups I and II were diabetic nephropathy (45.07% and 43.83%), hypertensive nephropathy (18.30% and 19.17%), chronic glomerulonephritis (11.26% and 9.58%), tubulointerstitial nephritis (8.45% and 6.84%), autosomal dominant polycystic kidney disease (4.22% and 5.47%), and unknown cause (12.67% and 15.06%). In the present study, the clinical features found in the patients at admission were anorexia, nausea, vomiting, weakness, weight loss, headache, pruritus, swelling over body, oliguria, anaemia, hypertension, and dyspnoea. The clinical features were almost similar at 0 week in both the groups. There was gradual improvement in clinical features in both the groups after 12 weeks of treatment but it was more marked in Rhubarb group. There was progressive decrease in both systolic and diastolic blood pressure towards normal in both the groups. As compared to control group, Rhubarb group showed significant (P < 0.05) reduction in both systolic and diastolic blood pressure after 12 weeks of treatment (Table 1). The total leucocyte count (TLC), differential leucocyte count (DLC), and platelet count remained within normal limits at the end of 12 weeks of treatment in both the groups. There was progressive improvement in various biochemical parameters in both the groups; Rhubarb group showed maximum improvement. As compared to control group, Rhubarb group showed significant increase in haemoglobin percent (P < 0.05), decrease in fasting and postprandial blood glucose (P < 0.01), decrease in blood urea (P < 0.05), and decrease in serum creatinine (P < 0.05) at 12 weeks. There was decrease in serum potassium in both the groups which was significant (P < 0.05) in Rhubarb group as compared to control. There was significant increase in serum calcium (P < 0.01), decrease in TUP (P < 0.05), increase in TUV (P < 0.001), and increase in GFR (P < 0.001) after 12 weeks of treatment in Rhubarb group as compared to control group (Table 2). The adverse drug reactions occurrence was not significantly different between control and Rhubarb groups. According to Modified Hartwig and Siegel Scale, the adverse drug reactions were mild (no hospitalization, no change of therapy, and no additional treatment) in severity in both the groups. No adverse event was of acute onset (within 60 minutes). On Naranjo's Scale, the ADRs were possible (scores = 1–4) in 12 cases and probable (scores = 5–8) in 11 cases with control group while possible (scores = 1–4) in 14 cases and probable (score = 5–8) in 8 cases with Rhubarb group (Table 3). BODY.4. DISCUSSION: Chronic kidney disease (CKD) is an emerging chronic disease globally due to rapidly increasing incidence of diabetes and hypertension worldwide [15, 16]. CKD leads to premature morbidity and mortality and hampers quality of life. In India, CKD is a major problem for both health sector and economy. More than 100,000 new patients enter RRT annually in India [17]. Because of meagre resources, only 10% of Indian ESRD patients receive any RRT. The monthly cost of hemodialysis is $300, whereas CAPD costs $600. The cost of transplant is $8900 in the first year, which declines later to $3000 annually. Among the RRT options, renal transplant is the preferred choice as it is cost effective and offers better quality of life but still only a fraction of Indians can afford it [17]. Conservative management is very important to prevent CKD and to prevent progression of CKD to ESRD. It delays the progressive deterioration of renal function. It provides only symptomatic relief. So, newer treatment modalities are being searched which can halt nephron damage, delay the development of ESRD and are cost effective. Previous studies have reported beneficial effect of rhubarb in CKD patients [18, 19]. Rhubarb contains various phytoconstituents among which rhein and emodin are important because of their beneficial effect in CKD. Rhein inhibit cell hypertrophy and extracellular matrix (ECM) accumulation by decreasing the transforming growth factor-beta 1 (TGF-β1) and fibronectin expression in renal tissue [20]. TGF-β1 stimulates the glucose uptake in mesangial cells through upregulation of GLUT 1 expression. Emodin has inhibitory effect on the expression of c-myc mRNA and hence on cell cycle downregulation in cultured rat mesangial cells, which might be the reason why emodin inhibits mesangial cell proliferation [21]. Rhubarb suppresses the production of various cytokines from macrophages and human mesangial cells [22, 23]. Rhubarb also has laxative effect which increases excretion of nitrogenous wastes from the body [7, 8]. These might be the probable mechanisms for beneficial effects of rhubarb in our study. Rhubarb showed beneficial effects in CKD patients at a dose of 1000 mg/day [19]. So, Rhubarb dose used in our study was 350 mg TDS daily. According to Ye et al., there was no side effect of rhubarb administration at a dose of 8–12 g/day for 3 weeks in 30 patients of CKD [18]. So, the ADRs might be the manifestations of underlying renal pathology or due to other coadministered drugs. The findings in our study are in accordance with those reported in previous studies. The drawback of this study is its limited duration of study. Longer duration of follow-up is needed in further studies to see the long term effect of rhubarb in CKD patients. So, supplementation of Rhubarb along with conservative management produces improvement in clinical features and in biochemical parameters and is safe in patients of chronic kidney disease. BODY.5. CONCLUSION: Rhubarb supplementation improved the therapeutic effect of conservative management in stage 3 and stage 4 patients of chronic kidney disease.

TITLE: Effectiveness of a Hospital-Based Work Support Intervention for Female Cancer Patients – A Multi-Centre Randomised Controlled TrialEffect of Hospital-Based Work Support Intervention ABSTRACT.OBJECTIVE: One key aspect of cancer survivorship is return-to-work. Unfortunately, many cancer survivors face problems upon their return-to-work. For that reason, we developed a hospital-based work support intervention aimed at enhancing return-to-work. We studied effectiveness of the intervention compared to usual care for female cancer patients in a multi-centre randomised controlled trial. ABSTRACT.METHODS: Breast and gynaecological cancer patients who were treated with curative intent and had paid work were randomised to the intervention group (n = 65) or control group (n = 68). The intervention involved patient education and support at the hospital and improvement of communication between treating and occupational physicians. In addition, we asked patient's occupational physician to organise a meeting with the patient and the supervisor to make a concrete gradual return-to-work plan. Outcomes at 12 months of follow-up included rate and time until return-to-work (full or partial), quality of life, work ability, work functioning, and lost productivity costs. Time until return-to-work was analyzed with Kaplan-Meier survival analysis. ABSTRACT.RESULTS: Return-to-work rates were 86% and 83% (p = 0.6) for the intervention group and control group when excluding 8 patients who died or with a life expectancy of months at follow-up. Median time from initial sick leave to partial return-to-work was 194 days (range 14–435) versus 192 days (range 82–465) (p = 0.90) with a hazard ratio of 1.03 (95% CI 0.64–1.6). Quality of life and work ability improved statistically over time but did not differ statistically between groups. Work functioning and costs did not differ statistically between groups. ABSTRACT.CONCLUSION: The intervention was easily implemented into usual psycho-oncological care and showed high return-to-work rates. We failed to show any differences between groups on return-to-work outcomes and quality of life scores. Further research is needed to study which aspects of the intervention are useful and which elements need improvement. ABSTRACT.TRIAL REGISTRATION: Nederlands Trial Register (NTR) 1658 BODY.INTRODUCTION: In recent years, advances in cancer screening and cancer treatment have improved the survival rates for patients with cancer. An increasing number of cancer patients are therefore able to live many years beyond the original cancer diagnosis and face new challenges upon cancer survivorship. For cancer patients of working age, returning to work is a key aspect of survivorship because it is often experienced as an important part of their recovery [1]. Furthermore, work contributes to personal, social, and economic well-being, and therefore return-to-work is associated with the quality of life of cancer patients [2]–[4]. Unfortunately, not all cancer patients are able to return-to-work and many of these patients have more adverse work outcomes in comparison to the general population. For instance, the risk of unemployment is estimated to be 37% higher for cancer patients compared to non-cancer controls [5] and return-to-work rates are estimated to vary between 30–93% depending on for instance cancer type and treatment [6], [7]. Furthermore, a portion of cancer patients face a decrease in income [8] and suffer from impaired work functioning compared to the general population [9], [10]. Finally, the employer and the society at large are also affected due to the costs of absenteeism, disability pension, and loss of productivity [11]. Intervention studies aimed at enhancing the return-to-work of cancer patients are rare, especially randomised controlled trials [12], [13]. However, we developed an intervention based on previous studies that demonstrated effective results for enhancing the return-to-work of cancer patients [12], and we developed this intervention together with various stakeholders involved in the return-to-work process of cancer patients [14]. An early intervention – meaning soon after diagnosis or early in treatment - is most appropriate because the longer the duration of sick leave, the more difficult return-to-work is to achieve [15]. For delivering an early intervention, a hospital-based intervention is most appropriate, as most cancer patients do not have contact with their employer or occupational physician during early phases of their cancer treatment and their advice seems to be influential [16], [17]. In addition, previous studies have shown that early interventions could be most effective [12]. Furthermore, return-to-work should be part of the complete psycho-oncological care package and should not be dealt with in isolation [18]. Our hypothesis is that a hospital-based intervention will enhance the return-to-work of cancer patients, as work is not typically addressed at the hospital [19]. Furthermore, an important and modifiable prognostic factor for the return-to-work of cancer patients is self-assessed work ability which varies according to treatment type and cancer diagnosis [7]. Self-assessed work ability may readily be improved by providing patient education and support that addresses misconceptions concerning return-to-work [20]. The objective of this study was to determine the effect of a hospital-based work support intervention for cancer patients on return-to-work and quality of life, which was achieved. BODY.MATERIALS AND METHODS: The medical ethics committee of the Academic Medical Center approved the study, and the medical ethics committees of the six participating hospital advised positively regarding feasibility of the study. Patients signed informed consent forms prior to participation in the study. This trial was registered at the Dutch National Registry:NTR1658 (http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=1658). Both the design of the study and the content of the hospital-based work support intervention have been described in detail elsewhere [14]. We used items from the CONSORT statement for improving the quality of reporting randomised trials [21]. The protocol for this trial and supporting CONSORT checklist is available as supporting information; see Checklist S1 and Protocol S1. BODY.PATIENTS: Cancer patients between 18 and 60 years of age who had been treated with curative intent at one of the six participating hospital departments, had paid work, and who were on sick leave were eligible to participate. Treatment with curative intent was defined as an expected 1-year survival rate of approximately 80%. We excluded patients who were not sufficiently able to speak, read, or write Dutch, had a severe mental disorder or other severe co-morbidity, and for whom the primary diagnosis of cancer had been made more than two months previously. We monitored non-response by assessing the proportion of patients who participated in comparison to all eligible patients. BODY.HOSPITAL-BASED WORK SUPPORT INTERVENTION: The hospital-based work support intervention began a few weeks after the onset of the study and was spread across a maximum of 14 months. The hospital-based work support intervention consisted of the following components: 1) delivering patient education and support at the hospital, as part of usual psycho-oncology care; 2) improving communication between the treating physician and the occupational physician; and 3) drawing-up a concrete and gradual return-to-work plan in collaboration with the cancer patient, the occupational physician, and the employer [14]. We integrated patient education and support regarding return-to-work into the usual psycho-oncological care in the form of 4 meetings that lasted 15 minutes each. This care was delivered by an oncology nurse or medical social worker (hereafter referred to as nurse). In addition, a least one letter was sent to the occupational physician to enhance communication. We also asked the occupational physicians to organise a meeting between the patient and the employer to draw-up a return-to-work plan. The key aspects of the hospital-based work support intervention included the patient education and support at the hospital and the provision of information to the occupational physician. In the Netherlands, patients must provide their consent to allow medical information to be sent from a treating physician to an occupational physician. Therefore, we were only able to inform the occupational physicians of patients who provided this form of consent. BODY.STUDY DESIGN: This study was designed as a multi-centre randomised controlled trial with a follow-up period of two years. Here we report the results of the first follow-up year. Eight departments from six hospitals in the Netherlands participated in the study. The treating physician or nurse informed the cancer patients of the study a few weeks after their diagnosis and determined patient eligibility by assessing the inclusion and exclusion criteria. The research team contacted patients who were eligible and willing to participate and enrolled these patients in the study. After the patients had filled in the baseline questionnaire, one of us [ST] allocated the eligible patients to the intervention or to the control group using the computerised randomisation programme ALEA [22]. The allocation ratio was set as equal in the programme. Stratified randomisation was applied for two important prognostic factors for return-to-work [23]; age (<50 or ≥50) and cancer diagnosis (i.e. hospital department). Minimisation was applied to equalise group sizes. The patient date of each consecutive patient were entered in the programme and according to the conditions mentioned above the programme randomly assigned the patients to the intervention or the control group. The allocation was irrevocable and was not changed during the study nor during the analysis. Patients and providers were immediately informed of the allocation as it was impossible to conceal allocation for this intervention. Questionnaires were administrated to the patients at baseline and at 6 and 12 months of follow-up. The follow-up questionnaires were mailed to the patients' homes with a postage-paid envelope enclosed. Both the questionnaire data and the information from the nurses who delivered the intervention were gathered for the economic evaluation. Outcome measures and cancer treatment were assessed at all time points. Socio-demographic factors and prognostic factors for time until return-to-work were assessed at baseline only. BODY.MEASUREMENTS: The primary outcomes were return-to-work and quality of life. The intervention was considered effective if patients in the intervention group had a significantly shorter time to return-to-work (in days) than did patients in the control group, provided that their quality of life had not significantly deteriorated. Return-to-work was measured both as the rate of return-to-work at one year of follow-up and as the number of calendar days between the first day of sick leave and the first day at work (either part-time or full-time) that was sustained for at least 4 weeks. Quality of life was assessed with the Short Form-36 (SF-36) [24], which included all subscales and a Visual Analogue Scale (VAS). Secondary outcomes included work ability, work functioning, and costs. Work ability was assessed using the first question of the Work Ability Index (WAI) [25]. Impaired work functioning was assessed with the Work Limitation Questionnaire (WLQ) [26], which could only be filled in if a patient had (partly) returned to work. We conducted the economic evaluation from a societal perspective. We included lost productivity costs and work adjustments costs for both groups and costs to deliver the intervention for the intervention group. Productivity loss was determined by multiplying the cumulative net number of hours on sick leave by the estimated price of productivity loss based on age and gender [27]. We assumed that when a patient partially returned to work, his/her productivity was 100% during the hours of partial work resumption. We calculated productivity losses using both the human capital approach and the friction costs approach [27]. For the human capital approach, all hours on sick leave were included for 100%. For the friction costs approach, all hours on sick leave with a maximum of 167 days were included for 80% [27]. Costs to deliver the intervention were determined by combining the training costs and the costs to deliver the intervention. Training costs consisted of trainer costs, study material costs, and attendance costs for the nurses. Costs to deliver the intervention consisted of the mean hour of investment multiplied by the average nurse wage and subsequently multiplied by 42% overhead costs [27], and the mean hour of investment of the secretary for sending of the letters to the occupational physician, as well as the printing costs for the information leaflet. As the letter from the treating physician to the occupational physician was a copy of the letter to the general practitioner, no additional costs for the treating physician to produce these letters were taken into account. The socio-demographic factors measured at baseline included the number of days between the first day of sick leave and enrolment in the study, marital status, time since diagnosis, breadwinner status, position at work, shift work, years in current position, years of paid employment, income, importance of work (VAS), and company size. Prognostic factors for time to return-to-work of the cancer patients included [7], [23] age, gender, education, diagnosis, cancer treatment, number of working hours according to contract, physical workload (Questionnaire of Perception and Judgement of Work (VBBA)) [28], fatigue (Multidimensional Fatigue Inventory (MFI)) [29], depression (Centre for Epidemiologic Studies for Depression Scale (CES-D)) [30], co-morbidity, self-efficacy (general self-efficacy scale (ALCOS)) [31], and clinical characteristics (i.e. diagnosis and treatment). BODY.SAMPLE SIZE: The calculation of the patient sample size was based on two earlier studies focused on return-to-work in cancer patients [23], [32]. Based on the return-to-work rates in these studies, we assumed a relative risk of not returning to work of 0.53 for individuals in the intervention group versus those receiving usual care [14]. With a power of 80% and two-sided significance level of p<0.05, the sample size required was 109 patients in each group [33]. Assuming that 20% of the initial patients would be lost to follow-up, 270 patients should have been recruited to gather 246 patients at 12 months of follow-up. To account for at least 10% missing data at baseline, 300 patients sought to be included in the study. BODY.STATISTICAL ANALYSIS: Data entry was verified by means of a 20% double data entry and a 100% double data check regarding the rate and time of patients until return-to-work. Participants who did and did not whish to participate were analysed on age using Student's t-test. All analyses were performed according to the intention-to-treat principle, which meant that all patients were included in the analysis. We censored patients who dropped out of the study because of missing data. Therefore, differences between patients who dropped out or completed the study were analysed according to their baseline quality of life scores. All data were analysed by means of descriptive statics using PASW version 18. The baseline data were assessed to evaluate whether there was an imbalance between the intervention group and the control group using Student's t-test for continuous variables and the χ2 test for categorical variables. We considered a p-value≤0.05 to be statistically significant. We calculated relative risks and 95% confidence interval for returning to work (full and partial) at 12 months of follow-up for the intervention group versus the control group. The median time until return-to-work was analysed with a Kaplan-Meier survival analysis, and differences between groups were tested with the log rank test. In addition, the Cox proportional hazard model of survival analysis was applied to estimate hazard ratios and the corresponding 95% confidence intervals for the time until return-to-work (full and partial) with a hazard ratio <1 indicating a longer time to return-to-work. Improvements in the subsequent primary outcome of quality of life and the secondary outcomes of work ability and work functioning between groups were examined using a longitudinal multilevel analysis. Mean costs between the groups were analysed using Student's t-test. BODY.RESULTS: Cancer patients who were diagnosed at one of the participating hospital departments between May 2009 and December 2010 and who were eligible and willing to participate were enrolled in the study. The enrolment of new patients ended in December 2010 to enable the inclusion of patient follow-up data within the time constraints of the study. A total of 755 of the 855 cancer patients were excluded; 611 did not meet the eligibility criteria primarily because they were too old, 119 declined participation, and 25 were excluded for other reasons, and this led to an overall response rate of 47% (Figure 1). In total, 133 cancer patients were included in the study; 65 were assigned to the intervention group and 68 were assigned to the control group. At baseline, all 133 patients provided complete data on the primary outcome, whereas 132 (99%) patients provided complete data on the secondary outcomes (Figure 1). The response rate at 12 months of follow-up was 128 (96%) for the outcome of return-to-work and was 108 (81%) for the outcome of quality of life and secondary outcomes. The reason why patients did not return the questionnaire included cancer recurrence (4 patients; 3%), decline (6;5%) or were unknown 11 (8%), while 4 (3%) patients died within the 12-months follow-up period (Figure 1). Patients were on average 47.5±7.9 years old. Breast cancer was the most common diagnosis (62%), followed by cancer diagnosis of the female reproductive system (34%) (Table 1). No statistically significant differences between the intervention group and the control group on any of the socio-demographic or prognostic characteristics measured at baseline or any medical characteristics measured at follow-up were identified (Table 1). 10.1371/journal.pone.0063271.g001Figure 1BODY.PATIENT FLOW.: 10.1371/journal.pone.0063271.t001Table 1BODY.PATIENT CHARACTERISTICS.: Patient characteristics Intervention group (N = 65) Control group (N = 68) P-value ** Socio-demographic characteristics * Age (years) ¥ 47.5±8.2 47.6±7.8 0.92 Gender (% female) 99% 100% 0.31 Marital status (% married or living with partner) 79% 69% 0.20 Breadwinner position (% sole or shared) 65% 56% 0.36 Education level (%) Low 11% 16% 0.53 Intermediate 59% 51% High 30% 33% Clinical characteristics * Diagnosis Breast cancer 64% 60% 0.82 (%) Cervix cancer 23% 22% Ovarian cancer 5% 10% Vulva cancer 3% 3% Other 5% 5% Number of co- 0 45% 54% 0.09 morbidities 1 22% 31% (%) ≥2 33% 15% Surgery (%) 99% 96% 0.78 Chemotherapy (%) 66% 71% 0.84 Radiotherapy (%) 60% 58% 0.67 Work-related characteristics * Type of occupation Health care/education 38% 37% 0.69 (%) Administrative 9% 9% Sales 5% 12% Other 48% 42% Type of work (% mainly physically work) 32% 40% 0.38 Physical workload (0–28) ** 4.7±3.6 5.7±4.4 0.18 Time since sick listed (days) 26.5±35.1 15.0±53.2 0.15 Importance of work (0–100) ** 58.7±23.1 51.5±28.3 0.11 Shift work (% shift work) 26% 19% 0.36 Type of contract Permanent 89% 84% 0.17 (%) Temporary 11% 9% Self-employed 0% 4% Other 0% 3% Health-related characteristics * Fatigue (MFI ** General fatigue (0–20) 12.4±4.9 13.1±4.3 0.37 Depression (CES-D) ** Sum score (0–60) 14.1±9.3 13.5±7.7 0.67 Self-efficacy (ALCOS) ** Sum score (0–80) 66.5±8.6 66.2±7.6 0.83 *Continuous variables: mean ± standard deviation; nominal and ordinal variables percentages. **Higher scores represent higher level of physical workload, importance of work, fatigue, feelings of depression, and self-efficacy. ¥Age at the time of randomisation. BODY.HOSPITAL-BASED WORK SUPPORT INTERVENTION: No harm or unintended effects were reported by patients as a result of participating in the intervention. Seven patients (12%) assigned to the intervention group did not receive the patient education and support from the nurse [34]. For all patients who provided this type of consent (86%), at least one letter from the treating physician was sent to the occupational physician. In five cases (10%), the patients' occupational physician organized a meeting between the patient, supervisor, and himself to draw-up a return-to-work plan. The median number of contacts made between the nurse and the patient was 4 (range 1–4) and the median duration of each meeting was 23 minutes (range 7–60). Eight (12%) patients assigned to the control group reported having received patient education or support regarding their return-to-work from their nurse. BODY.PRIMARY OUTCOME – RETURN-TO-WORK AND QUALITY OF LIFE: The return-to-work rate (full or partial) of all 128 randomised patients with follow-up data at 12 months was 79% for the intervention group and 79% for the control group (p = 0.97), and these rates were 86% and 83%, respectively (p = 0.61), when patients who died within the follow-up period or those with a life expectancy of only a few months were excluded. The relative risk of returning to work (full or partial) for the intervention group versus the control group was 1.03 (95% CI 0.84–1.2). Of the patients who did not return-to-work (intervention versus control group); 2 versus 2 died, 3 versus 1 had a life expectancy of few months, 4 versus 5 lost their jobs, 2 versus 5 experienced adverse side-effects, and 2 versus 0 had other reasons. Median time from the initial sick leave until partial return-to-work was 194 days (range 14–435) for the intervention group and 192 days (range 82–465) for the control group (log rank test; p = 0.90). Median time from initial sick leave until full return-to-work was 283 days (range 25–394) for the intervention group and 239 days (range 77–454) for the control group (log rank test; p = 0.52). Figure 2 summarizes Kaplan-Meier survival analyses for the two groups on partial and full return-to-work. The hazard ratio for partial return-to-work was 1.03 (95% CI 0.64–1.6) for the intervention group versus the control group and was 0.88 (95% CI 0.53–1.5) regarding full return-to-work. 10.1371/journal.pone.0063271.g002Figure 2BODY.KAPLAN-MEIER SURVIVAL CURVES FOR TIME TO PARTIAL RETURN-TO-WORK (A) AND FULL RETURN-TO-WORK (B).: Quality of life scores showed statistically significant improvements over time (p ranged between 0.014 to ≤0.001) but did not differ statistically significant between groups (p ranged between 0.15 to 0.99) (Table 2). 10.1371/journal.pone.0063271.t002Table 2BODY.QUALITY OF LIFE, WORK ABILITY, AND WORK FUNCTIONING.: Group Baseline 6 months follow-up 12 months follow-up P-value** Quality of life* (SF- Physical functioning Intervention 76±28 71±21 81±16 0.95 36) (0–100) Control 73±28 70±22 79±20 (N = 133) Role-physical Intervention 48±44 29±40 47±40 0.46 Control 50±43 31±37 61±41 Vitality Intervention 60±21 51±20 59±19 0.60 Control 57±17 51±16 56±16 General health Intervention 61±21 54±18 64±17 0.15 Control 61±18 59±18 70±19 Social functioning Intervention 70±23 66±24 75±20 0.46 Control 68±22 66±22 78±20 Role-emotional Intervention 49±44 53±45 64±42 0.71 Control 52±41 64±44 71±40 Mental health Intervention 65±17 71±16 77±15 0.32 Control 64±16 70±16 72±15 Pain Intervention 69±30 67±25 75±21 0.99 Control 70±23 69±20 76±17 Quality of life* Intervention 60±22 62±23 73±17 0.26 VAS (0–100) (N = 133) Control 61±21 67±18 70±17 Overall work ability* Intervention 5±3 4±3 6±2 0.59 (WAI) (0–10) (N = 133) Control 5±3 5±3 7±2 Overall work productivity* Intervention NA 34±19 29±15 0.68 (WLQ) (0–100) (N = 100) Control NA 30±14 27±16 Mean ± sd; *Higher scores represent a higher level of functioning/well-being/quality of life, work ability, and work functioning. **P-value represents the interaction effect of time and group. BODY.SECONDARY OUTCOMES – WORK ABILITY, WORK PRODUCTIVITY, AND COSTS: Work ability improved statistically significant over time (p≤0.001) but did not differ statistically significant between groups (p = 0.58) (Table 2). Work functioning did not improve significantly over time (p = 0.3) and did not differ significantly between groups (p = 0.48) (Table 2). Table 3 shows that the intervention costs were 119 Euros per patient in the intervention group. The mean (± SD) lost productivity cost according to the human capital approach was 41.393 (±39.269) Euros in the intervention group and 38.968 (±38.399) Euros in the control group. The mean (± SD) lost productivity cost according to the friction costs approach was 14.030 (±3.614) Euros in the intervention group and 13.529 (±3.313) Euros in the control group. The mean work accommodations cost was 2.975 and 3.025 Euros in the intervention group and control group, respectively. These costs did not differ statistically between groups. 10.1371/journal.pone.0063271.t003Table 3BODY.ECONOMIC EVALUATION.: Costs of the work-directed intervention in Euros Description Costs (€) Training costs 1 trainer, time investment 24 hours, 50 Euros per hour 1200 Study material, refreshments 125 Attendance costs nurses, 11 nurses, 30 Euros per hours, 4 hours 1320 Total training costs per patient in the intervention group 41 Work-directed intervention Mean hour of investment of nurse was 1.2 hour, 43 Euros per hour 66 Mean hour of investment of secretary was 0.16 hour, 30 Euros per hour 5 Informational leaflet 7 Total intervention costs per patient in the intervention group 78 Total costs per patient in the intervention group 119 Costs differences between groups in Euros N Intervention group Control group Mean difference P-value Productivity loss net HCA Mean (±SD) Euros 128 41393 (±39269) 38968 (±38399) −2425 0.72 Productivity loss net FCA 128 14030 (±3614) 13529 (±3313) −438 0.48 Mean (±SD) Euros Work adjustments 3* 2975 3025 (±71) 50 0.67 Mean (±SD) Euros Work-directed intervention Mean Euros 128 119 0 −119 NA Abbreviations: HCA Human Capital Approach; FCA Friction Costs Approach; NA: not applicable; SD = standard deviation. *Only three patients had work adjustments that were not related to productivity. BODY.DISCUSSION: The objective of this study was to determine the effect of a hospital-based work support intervention for female cancer patients on return-to-work and quality of life. In general, return-to-work rates were high. We failed to show any differences between groups on return-to-work outcomes and quality of life scores. BODY.STRENGTHS AND LIMITATIONS: One strength of our study was the innovative approach that was used to address the adverse work outcomes of cancer patients. Few studies have addressed this important subject by developing an intervention that is primarily aimed at enhancing the return-to-work of cancer patients [12], [13], [34]. Furthermore, another strength of this study was the use of a low-cost intervention that could be implemented without substantially increasing the time required, which is important because of the burden on cancer care. In addition, this intervention was easily adapted to the existing variation in usual psycho-oncological care, which yields high external validity. One limitation of our study was the inability to include sufficient patients, according to our predetermined power analysis, which led to greater uncertainty in the results. BODY.INTERPRETATION OF FINDINGS: We found that the intervention was easily accepted in usual psycho-oncological care and we found that patients were notably satisfied with the intervention [34]. For those reasons, addressing the return to work of cancer patients is highly relevant for usual psycho-oncological care. We found similar return-to-work outcomes and quality of life scores for both groups. There are several possible explanations for the lack of statistically significant difference between groups, which can be sought in the intervention content and the study design. The basic assumption behind the intervention was that return to work would increase by means of improved self-assessed work ability as a result of patient education and support that addressed misconceptions about cancer and work. We found that self-assessed work ability increased significantly over time but did not differ significantly between groups. It is possible that addressing these misconceptions could have required a more intense intervention or that the training we provided to the nurses was not sufficient. We do not know precisely which misconceptions impede return to work and which should be addressed. On the other hand, this later possibility was indicated as a number of nurses mentioned that they were not completely convinced of their competence to deliver the return-to-work advice. It may be that our half-day training course was too short to enable nurses to gain the knowledge required to adequately address patients' misconceptions about return to work adequately. For these reasons, it is possible that certain misconceptions regarding cancer and work could have persisted and may have resulted in the absence of an intervention effect. In addition, we experienced difficulties in involving the occupational physician and the employer for the intervention while their involvement appeared to be important [35] and may have caused the absence of an intervention effect. There are some observational studies that showed that the treating physician's advice about return-to-work influenced work resumption considerably either with a shorter or with a longer return-to-work [16], [17], [34]. However, our study shows that apparently this is an overestimation that is not reproduced in an experimental study. BODY.METHODOLOGICAL CONSIDERATIONS: In accordance with the intention to treat analysis we included in the survival analysis patients who died within the follow-up period as censored. However, an assumption in survival analysis is that when a patient is censored, the change that a patient will be able to achieve the outcome is still 50% [36], [37], which is not the case in this situation. However, on a population of 133 patients, we do not expect that the 4 patients who were equally divided between the intervention group and control group, influenced the findings significantly. BODY.EXTERNAL VALIDITY: It is generally acknowledged that the disability legalisation of a country influences the return-to-work outcomes of employees on sick leave and that disability legalisations varies widely among countries [38]. For that reason, the effect of interventions on return-to-work may also vary from one country to another. The results and conclusions of this study are relevant for the Netherlands due to its social security legislation. However, the early hospital-based work support intervention integrated into usual psycho-oncological care could be adapted and generalised to other countries because cancer patients in other countries experiencing a lack of support about their return-to-work from the hospital as often as patients in the Netherlands [39]. The exact content of the intervention should be adapted to the social security legislation of the country it is implemented in. BODY.RECOMMENDATIONS FOR FURTHER RESEARCH AND PRACTICE: In terms of recommendations for clinical practice, this study revealed that psycho-oncological care can address the return-to-work of cancer patients early in their treatment, as well as follow-up, as the intervention was appreciated by patients and was perceived as useful and feasible by the nurses. Since, the occupational physician and employer involvement is pivotal for a successful return to work but appeared problematic in our intervention, it seems important to solve what impedes their involvement and to adapt the intervention accordingly. As we found similar work outcomes between the intervention group and the control group, a recommendation for further research is to study if an improved intervention leads to shorter time to return-to-work. Due to the large range in time to return to work, it seems important to identify patients who have a higher risk of getting adverse work outcomes based on a clinical prediction rule. Therefore, a recommendation for further research is, to develop such a clinical prediction rule for work outcomes and to evaluate it for the accuracy in identifying patients with a higher risk of adverse work outcomes. Furthermore, apart from identifying patients with a higher risk, it is also important to make the intervention more tailored as it appeared that some patients do not need an intervention to achieve a successful return to work while other patients might have benefited from a more intense intervention. A possibility to make an intervention more tailored is by using a stepped care model. This means that a low-intensity intervention can be offered to all patients while a high-intensity intervention is only offered to patients, for whom work resumption turns out to be problematic. We found that the contrast between groups was reduced, due to the study design. Therefore, another recommendation for further research would be to consider alternative study designs, such as a cluster randomised controlled trial [40]. BODY.SUPPORTING INFORMATION: Checklist S1 CONSORT Checklist. (DOC) Click here for additional data file. Protocol S1 Trial Protocol. (PDF) Click here for additional data file.

TITLE: Beta-agonist overuse and delay in obtaining medical review in high risk asthma: a secondary analysis of data from a randomised controlled trial Asthma mortality surveys report delays in seeking medical review and overuse of beta-agonist therapy as factors contributing to a fatal outcome. However, the strength of these associations is limited because many asthma deaths are unwitnessed. We undertook a secondary analysis of data from a 24-week randomised controlled trial of 303 patients with high-risk asthma, randomised to combination budesonide/formoterol inhaler according to a single maintenance and reliever therapy regimen or fixed dose budesonide/formoterol with salbutamol as reliever (Standard) regimen. Medication use was measured by electronic monitors. The thresholds for high, marked and extreme beta-agonist use days were defined in the single maintenance and reliever therapy arm as: >8, >12 and >16 actuations of budesonide/formoterol in excess of four maintenance doses, respectively; and in the Standard arm as: >16, >24 and >32 actuations of salbutamol, respectively. Whether a medical review was obtained within 48 h of an overuse episode was determined by review of data collected during the study by participant report. The mean (standard deviation) proportion of days in which high, marked and extreme beta-agonist overuse occurred without medical review within 48 h was 0·94(0·20), 0·94(0·15) and 0·94(0·17), and 0·92(0·19), 0·90(0·26) and 0·94(0·15) for single maintenance and reliever therapy and Standard regimens, respectively. In at least 90% of days, in which beta-agonist overuse occurred, patients did not obtain medical review within 48 h of beta-agonist overuse, regardless of the magnitude of overuse or the inhaled corticosteroid/long-acting beta-agonist regimen. ABSTRACT.RELIEVER INHALER OVERUSE AND DELAY IN MEDICAL REVIEW IN ASTHMA: In asthma, overuse of beta-agonist reliever medication and delay in seeking medical review in an exacerbation are linked to asthma deaths. Janine Pilcher at the Medical Research Institute of New Zealand, and co-workers, conducted a review of data from a study of 303 adult patients with severe asthma, followed over 24 weeks. The patients were allocated to either a budesonide/formoterol, or a salbutamol inhaler to take for symptom relief, in addition to their maintenance treatment. Inhalers were fitted with electronic monitors, to accurately document every use. In both groups, on 90% of days when an exacerbation requiring excess use of an inhaler occurred, patients did not follow-up with medical professionals within 48 h as advised. Further, in both groups, ‘extreme’ reliever inhaler use was recorded at least once in around one in four patients. ABSTRACT.INTRODUCTION: Asthma mortality surveys report that delay in seeking medical review is a common and important factor contributing to a fatal outcome.1–5 Beta-agonist overuse immediately before asthma death has also been reported, but the strength of this association is limited because these surveys rely on reports from relatives and friends of patients who have died from asthma.1–5 Furthermore, many asthma deaths are unwitnessed. Prescription database surveys identify a strong association between increased dispensing of short-acting beta-agonist drugs and the risk of mortality.6–10 The risk of asthma death increases markedly when more than 1.4 inhaler canisters per month are dispensed.7 Although this is estimated to be equivalent to an average of ten actuations per day, this may not correspond to actual patterns of use during a severe exacerbation. Patients with asthma also often report taking very high doses of beta-agonists during severe exacerbations leading to hospital admission, but these estimates may be influenced by recall bias.11 In the situation of stable asthma, patients who overuse their medications are likely to under-report actual use and conversely those who underuse tend to over-report actual use.12 The best method to assess actual patient self-administration of beta-agonist is to use validated electronic monitors that record the exact date and time of each actuation.13, 14 This approach has been used in one 13-week observational study in children15 and also in one 24-week randomised controlled trial (RCT) of two regimens based on maintenance combination budesonide/formoterol inhaler therapy in high risk adult asthma.16 In this RCT, we reported long-term patterns of beta-agonist use16 as well as use during severe exacerbations leading to hospital attendance.17 We identified that in less than 10% of days when high beta-agonist use occurred, defined as > 16 actuations of salbutamol (or equivalent) in a 24-h period, did patients obtain medical review within 48 h, despite this advice being verbally explained and documented in the asthma action plans provided.16 About one in four patients self-administered >32 actuations per day of salbutamol (or equivalent) on at least one occasion during the study. These findings showed the extent of unsupervised beta-agonist overuse by patients at risk of severe asthma exacerbations. In a subsequent analysis, we identified that very high doses of beta-agonist were commonly self-administered by patients without seeking medical review within 2 weeks before hospital presentation with a severe exacerbation of asthma.17 The objectives of this current analysis were to investigate the incidence of delay in obtaining medical review at different levels of excessive beta-agonist use throughout the 24 weeks of the RCT, and to describe patterns of beta-agonist use before and after severe exacerbations requiring systemic corticosteroid therapy. Our hypothesis was that most episodes of beta-agonist use, in excess of the thresholds at which medical review was recommended in the implemented asthma action plans, would not result in patients obtaining medical care. ABSTRACT.RESULTS: The participants in the RCT are described in Table 1. There were 151 participants randomised to the single maintenance and reliever therapy (SMART) regimen and 152 participants to the Standard regimen.Table 1Participant characteristics CharacteristicSMART group (N = 151)Standard group (N = 152)Mean [(standard deviation (SD)]Age (years) 41·3 (13·7) 42·6 (14·5) ACQ-5 Score 1·78 (0·99) 1·79 (2·4) Daily ICS dose (budesonide or equivalent), µg 805 (353) 813 (370) On-treatment FEV1 (L) 2·62 (0·91) 2·50 (0·78) On-treatment FEV1 % predicted 81·6 (18·9) 80·4 (20·5) Severe exacerbations in the prior 12 months 1·55 (1·31) 1·73 (1·22) Median (IQR) Baseline self-reported reliever usea 2 (1 to 3) 2 (1 to 3) Number of hospital admissions ever for asthma 1 (0 to 4) 1 (0 to 4) N/151 (%) N/152 (%) Male 48 (31·8) 46 (30·3)  Ethnicity   European 113 (74·8) 118 (77·6)   Māori 25 (16·6) 19 (12·5)   Pacific Islander 5 (3·3) 10 (6·6)   Other 8 (5·3) 5 (3·3) Number of severe exacerbations in previous 12 months  0 14 (9·3) 11 (7·2)  1 86 (57·0) 75 (49·3)  2 29 (19·2) 31 (20·4)  3 10 (6·6) 22 (14·5)  4 5 (3·3) 6 (3·9)  ≥5 7 (4·6) 7 (4·6) LABA use 92 (60·9) 103 (67·8) Combination ICS plus LABA inhaler 73 (48%) 82 (54%) Use of written asthma self-management plan 15 (10%) 20 (13%) Current smokers 30 (19·9) 29 (19·1) Ex-smokers 49 (32·5) 48 (31·6) Non-smokers 72 (47·7) 75 (49·3) ACQ asthma control questionnaire, FEV 1 forced expiratory volume in one second, ICS inhaled corticosteroids, LABA long acting beta-agonist a As per the ACQ question 6. ACQ question 6 is a categorical score of beta-agonist use over the preceding 7 days in the following bands: score 0—none; score 1—1 to 2 salbutamol inhalations most days; score 2—3 to 4 salbutamol inhalations most days; score 3—5 to 8 salbutamol inhalations most days; score 4—9 to 12 salbutamol inhalations most days; score 5—13 to 16 salbutamol inhalations most days; score 6—more than 16 salbutamol inhalations most days. ABSTRACT.HIGH, MARKED AND EXTREME BETA-AGONIST USE EPISODES WITHOUT MEDICAL REVIEW WITHIN 48 H: The proportions of participants with at least one high, marked and extreme use episode were similar in each regimen (Tables 2a–c, respectively). The total number of days of high, marked and extreme use were greater in the Standard regimen (Tables 2a –c, respectively).Table 2aHigh beta-agonist use without medical review OutcomeSMART group (N = 151)Standard group (N = 152)Relative risk or rate SMART vs Standard (95% CI) P N (%)Mean (SD)Median (IQR)Range N (%)Mean (SD)Median (IQR)RangeRiskRateAt least one episode of high use 84 (56) 68 (45) 1·24 (0·99–1·56) – 0·058 Number of days of high use 5·1 (14·3) 1 (0–3) 0–130 8·9 (20·9) 0 (0–5) 0–149 – 0·58 (0·39–0·88) 0·010 Number of days of high use in participants with at least one high use episode 9·1 (18·2) 2 (1–7·5) 1–130 19·9 (27·7) 7 (2–26·5) 1–149 – – – Number of days of high use without medical review within 48 h in participants with at least one high use episode 8·5 (17·8) 2 (1–6·5) 0–130 18·3 (24·8) 5 (2–26) 1–123 – 0·49 (0·31–0·75) 0·001 Proportion of high overuse days without medical review within 48 h compared to total high overuse days in participants with at least one high use episode 0·94 (0·20) 1 (1–1) 0–1 0·94 (0·15) 1 (1–1) 0·25 –1 – – – Table 2bMarked beta-agonist overuse without medical review OutcomeSMART group (N = 151)Standard group (N = 152)Relative risk or rate SMART vs Standard (95% CI) P N (%)Mean (SD)Median (IQR)RangeN (%)Mean (SD)Median (IQR)RangeRiskRateAt least one episode of marked overuse 54 (36) 56 (37) 0·97 (0·72–1·31) – 0·85 Number of days of marked overuse 2·6 (10·2) 0 (0–1) 0–109 4·8 (14·9) 0 (0–2) 0 to 144 – 0·56 (0·35–0·88) 0·013 Number of days of marked overuse in participants with at least one marked overuse episode 7·4 (16·0) 2 (1–7) 1–109 13·1 (22·3) 4·5 (2 to 15) 1–144 – – – Number of days of marked overuse without medical review within 48 h in participants with at least one marked overuse episode 6·7 (15·7) 2 (1–6) 1–109 11·7 (19·0) 4 (2–14) 0–118 – 0·62 (0·37–1·06) 0·079 Proportion of marked overuse days without medical review within 48 h compared to total marked overuse days in participants with at least one high use episode 0·94 (0·17) 1 (0·74–1) 0·15–1 0·92 (0·19) 1 (0·96–1) 0–1 – – – Table 2cExtreme beta-agonist overuse without medical review OutcomeSMART group (N = 151)Standard group (N = 152)Relative risk or rate SMART vs Standard (95% CI) P N (%)Mean (SD)Median (IQR)RangeN (%)Mean (SD)Median (IQR)RangeRiskRateAt least one episode of extreme overuse 41 (27) 40 (26) 1·03 (0·71–1·50) – 0·87 Number of days of extreme overuse 1·6 (6·7) 0 (0–1) 0–75 2·9 (12·2) 0 (0–1) 0–137 – 0·56 (0·34–0·91) 0·02 Number of days of extreme overuse in participants with at least one extreme overuse episode 5·8 (11·9) 2 (1–6) 1–75 11·0 (22·1) 4·5 (2·5–11) 1–137 – – – Number of days of extreme overuse without medical review within 48 h in participants with at least one extreme overuse episode 5·2 (11·9) 2 (1–4) 0–75 9·6 (18·3) 4·5 (2–10) 1–112 – 0·59 (0·31–1·10) 0·096 Proportion of extreme overuse days without medical review within 48 h compared to total extreme overuse days in participants with at least one high use episode 0·90 (0·26) 1 (1–1) 0–1 0·94 (0·15) 1 (1–1) 0·29–1 – – – In the SMART regimen, the number of high use days without medical review within 48 h was approximately half of that in the Standard regimen; relative rate (95% CI) 0·49 (0·31 to 0·75), P = 0·001 (Fig. 1, Table 2a). The point estimate of association was consistent with a lower number of marked and extreme use days without medical review within 48 h with the SMART regimen (Fig. 1 and Tables 2b, 2c, respectively).Fig. 1 a Mean number of high use days in participants with at least one high use episode, and mean number of high use days without medical review within 48 h in participants with at least one high use episode. b Mean number of marked overuse days in participants with at least one marked overuse episode, and mean number of marked overuse days without medical review within 48 h in participants with at least one marked overuse episode. c Mean number of extreme overuse days in participants with at least one extreme overuse episode, and mean number of extreme overuse days without medical review within 48 h in participants with at least one extreme overuse episode In both the SMART and Standard regimens, the proportion of high, marked and extreme use days without medical review within 48 h, compared to total days of high, marked and extreme use was at least 90% (Tables 2a, 2b and 2c, respectively). ABSTRACT.SEVERE EXACERBATIONS OF ASTHMA: During the study period, at least one severe asthma exacerbation was experienced by 28/151 (18·5%) of SMART participants and 50/152 (32·9%) of Standard participants. There was a total of 35 and 66 severe exacerbations in the SMART and Standard groups respectively; relative rate (95% CI) 0·54 (0·36 to 0·82), P = 0·004. ABSTRACT.DAILY MEDICATION USE PRIOR TO AND FOLLOWING SEVERE EXACERBATIONS: Medication use in the 14 days prior to and 14 days following a severe exacerbation for the SMART and Standard regimens is shown in Figs. 2 and 3, respectively. With the SMART regimen, there was increasing budesonide/formoterol use from about 5 days prior to initiation of oral corticosteroid therapy, with high use for a further 5 days before returning to baseline levels after 10 days. With the Standard regimen, there was variable salbutamol use from about 14 days prior to initiation of oral corticosteroid therapy, increasing markedly the day before, with high use for a further 3 days before returning to baseline levels after 10 days. In the severe exacerbation dataset, only one of the included days, 2 days prior to severe exacerbation for a patient in the Standard group, met dose dumping criteria, defined as greater than or equal to 100 actuations in a 3-h period.18 Fig. 2Median [interquartile range (IQR)] daily budesonide/formoterol actuations in the 14 days prior to, and the 14 days following a severe exacerbation in the SMART group. Day 0 represents the initiation of corticosteroid therapy. There were 35 severe exacerbations in the SMART group. For patients with repeat exacerbations, a minimal interval of 28 days between exacerbations was required, to avoid overlap of data within the same participant. One patient in the SMART group had a second severe exacerbation within 28 days following the prior exacerbation. Thus, electronic data for 34 severe exacerbations were included in the plots of medication use patterns for the SMART group Fig. 3Median (IQR) daily salbutamol a and budesonide/formoterol b actuations in the 14 days prior to, and the 14 days following a severe exacerbation in the Standard group. Day 0 represents the initiation of corticosteroid therapy. There were 66 severe exacerbations in the Standard group. For patients with repeat exacerbations, a minimal interval of 28 days between exacerbations was required, to avoid overlap of data within the same participant. Three patients in the Standard group had repeat severe exacerbations occurring within 28 days following the prior exacerbation. In another patient randomised to Standard treatment, there was no recorded data for two severe exacerbations due to use of non-study inhaled medication. Thus, electronic data for 61 severe exacerbations were included in the plots of medication use patterns in the Standard group. For these plots, only one of the included days (2 days prior to severe exacerbation for a patient in the Standard group) met dose dumping criteria (≥100 actuations in a 3 h period) The number of days in which high, marked or extreme use occurred in the 14 days prior to a severe exacerbation, expressed as a percentage of all high, marked and extreme use days was 56/766 (7%), 28/400 (7%) and 10/237 (4%) for SMART, and 166/1355 (12%), 111/735 (15%) and 80/438 (18%) for Standard, respectively. Of the high use episodes in the 14-day period prior to a severe exacerbation, 38/56 (68%) and 133/166 (80%) were not associated with medical review within 48 h, in the SMART and Standard regimens, respectively. Of the marked overuse episodes in the 14-day period prior to a severe exacerbation, 18/28 (64%) and 87/111 (78%) were not associated with medical review within 48  ABSTRACT.HIGHEST MEDICATION USE PRIOR TO AND FOLLOWING SEVERE EXACERBATIONS: In the SMART group, the median (range) highest daily number of budesonide/formoterol actuations was 11 (4 to 63) over the 14 days before severe exacerbations. In the Standard group, the median (range) highest daily number of salbutamol actuations was 13 (0 to 150) over the 14 days before severe exacerbations; on the day of highest salbutamol use preceding severe exacerbations, the median (range) number of budesonide/formoterol actuations was 4 (0 to 84). On this day of maximal salbutamol use, there were 8/61 (13%) instances when >8 actuations of budesonide/formoterol were taken and 10/61 (16%) instances when zero actuations of budesonide/formoterol were taken. In the SMART group, the median (range) highest daily number of budesonide/formoterol actuations was 10 (0 to 71) in the 14 days after oral corticosteroid use for severe exacerbations (Day 0). In the Standard group, the median (range) highest daily number of salbutamol actuations was 20 (0 to 152) in the 14 days after Day 0; on the day of highest salbutamol use following Day 0, the median (range) number of budesonide/formoterol actuations was 4 (0 to 33). On this day of maximal salbutamol use, there were 7/61 (11%) instances when >8 actuations of budesonide/formoterol were taken and 9/61 (15%) instances when zero actuations of budesonide/formoterol were taken. ABSTRACT.DISCUSSION: This study shows that in a high-risk population of adult patients with asthma, around 90% of beta-agonist overuse episodes, well in excess of the asthma action plan thresholds recommending prompt medical review, do not lead to patients obtaining medical review within 48 h. It is likely that the symptomatic relief obtained from the high doses of beta-agonist may be one of the underlying causes for the delay in or lack of seeking medical help in the situation of such poorly controlled, worsening asthma. We observed that in around 90% of marked and extreme overuse episodes, patients did not obtain medical review within 48 h despite this use being at least 1·5 and 2-fold higher than the level of use at which their asthma action plan advised that medical care must be sought. These findings complement our previous observation that 93% of high-use episodes, in which patients took at least 16 actuations of salbutamol or equivalent in a 24-h period, occurred without medical review within the next 48 h, despite this advice being documented in the provided asthma action plans.16 These findings were unexpected as they suggest that increasing beta-agonist use from at least 16 to at least 32 actuations of salbutamol or equivalent in a 24-h period did not lead to a greater likelihood of a patient seeking medical care. Patterns of beta-agonist use were also examined in the patients in whom a severe exacerbation was confirmed with the prescription of systemic corticosteroids. In the SMART regimen, the mean daily use of budesonide/formoterol progressively increased 5 days before the index corticosteroid use and remained high for a further 5-day period before returning close to baseline levels after 10 days. For the Standard regimen, the salbutamol use was variable from about 14 days prior to the index course of corticosteroids, increasing markedly the day before, with the high level of use gradually returning to baseline levels over 10 days. The majority of overuse episodes in the 14-day period prior to the initiation of oral corticosteroid therapy were not associated with medical review within 48 h, regardless of the magnitude of the overuse. These findings suggest that even in patients who did seek medical review, there is often delay during worsening asthma, resulting in the later prescription of oral corticosteroids in a severe exacerbation. This may have contributed to our finding that in the setting of a severe exacerbation it takes at least 5 days of oral corticosteroid treatment before there is sufficient improvement to result in a reduction in beta-agonist use, regardless of whether the SMART or Standard regimen was used. Different patterns of budesonide/formoterol use were observed in the Standard group during severe exacerbations. Some patients used no budesonide/formoterol on the day of highest salbutamol use preceding the severe exacerbation. This non-adherence may contribute to progressive worsening of unstable asthma, increasing the risk of a severe exacerbation. Another pattern was the use of budesonide/formoterol as an as required ‘reliever’ medication well in excess of the maintenance four actuations per day. These observations suggest that some patients may use their maintenance budesonide/ formoterol inhaler according to presumed need, similar to the SMART regimen, despite being prescribed fixed dose maintenance treatment and salbutamol for reliever. Another important observation was that while the SMART regimen reduced the number of high, marked and extreme overuse episodes by at least 40%, it did not influence the likelihood of a patient obtaining medical review when an overuse episode occurred. As a result, the SMART regimen reduced the total number of overuse episodes in which patients did not obtain medical review within 48 h, to a similar extent to which it reduced the total number of overuse episodes. Together with the 46% reduction in severe exacerbations, these findings suggest that the SMART regimen has a better safety profile as it reduced the total number of overuse episodes in which delay in obtaining medical review occurred. There are a number of methodological issues that require consideration. As an open-label study, there was the potential for bias, however the use of a single inhaler in the SMART regimen and separate inhalers in the Standard regimen without the requirement for dummy placebo inhalers enhanced generalisability of the findings to real world clinical practice. Metered dose inhaler (MDI)’s were used rather than dry powder inhalers (Turbuhaler) to deliver budesonide/formoterol, as it enabled us to use validated electronic monitors of inhaler use.19, 20 Beta-agonist overuse related to the measures of budesonide/formoterol and salbutamol for the SMART and Standard regimens, respectively. This is likely to have led to an underestimation of the number of beta-agonist overuse episodes for the Standard regimen, as it did not take into account the use of budesonide/formoterol as a reliever additional to the four maintenance actuations per day, a behaviour which was noted in some patients.17 There are several additional clinical implications of these findings. Firstly, the patient’s failure to recognise that the need for frequent and high doses of beta-agonist indicates severe asthma, likely contributes to the delay in or lack of seeking medical help. Secondly, this behaviour may reflect previous experience of these high risk patients with asthma, that most exacerbations of asthma requiring high doses of beta-agonist are self-limiting and eventually resolve without medical review, oral corticosteroids, or hospital attendance. This interpretation is supported by our observation that over 80% of all days of beta-agonist overuse, regardless of extent, did not lead to a course of oral steroids within the following 14 days. The higher proportions associated with the SMART regimen suggest that budesonide/formoterol overuse may be more likely to lead to resolution of the severe asthma episode without oral steroids than salbutamol overuse. Thirdly, even in the setting of an RCT with repeat clinic visits during which the asthma action plan was introduced and reinforced, most patients did not follow this written advice to obtain medical review when beta agonist use exceeded predefined levels of increased use. In conclusion, in at least 90% of days in which beta-agonist overuse occurred, patients with high-risk asthma did not obtain medical review within 48 h, regardless of the magnitude of the beta-agonist overuse, or the inhaled corticosteroid/long-acting beta-agonist regimen. ABSTRACT.METHODS: ABSTRACT.DESIGN: This is a secondary analysis of data from a 24-week multicentre, open-label RCT of Single combination budesonide/formoterol inhaler as Maintenance And Reliever Therapy (SMART regimen) vs. fixed-dose budesonide/formoterol with salbutamol for relief (Standard regimen) in 303 adult patients with a physician’s diagnosis of asthma, described previously.16 Patients were eligible if they had a current prescription for inhaled corticosteroids (ICS) and at least one asthma exacerbation in the preceding year. An exacerbation was defined as presentation to a General Practice or Emergency Department resulting in prescription of oral corticosteroids or treatment with spacer-delivered or nebulised bronchodilator, or self-administration of prednisone for asthma for at least 3 days. Exclusion criteria included a diagnosis of chronic obstructive pulmonary disease and current or ex-smokers with a > 10 pack-year smoking history with onset of respiratory symptoms after the age of 40. Patients were randomised to the SMART regimen: a MDI containing 200/6 µg budesonide/formoterol (Vannair, AstraZeneca Limited, Auckland, New Zealand, which is the MDI formulation of Symbicort turbuhaler), two actuations twice daily for maintenance with one extra dose as needed for relief; or the Standard regimen: 200/6 µg Vannair MDI, two actuations twice daily for maintenance with 100 µg salbutamol MDI (Ventolin, GlaxoSmithKline Limited, Auckland, New Zealand), one to two extra actuations for relief. All participants were given written asthma action plans and verbal instructions outlining when to consult their doctor. All participants provided written informed consent and the trial was approved by the New Zealand Multi Region Ethics Committee, and prospectively registered [ACTRN 12610000515099]. The study protocol is available at http://www.mrinz.ac.nz/uploads/mrinz/SMART_Protocol.pdf All Vannair and Ventolin MDIs incorporated an electronic monitor (Smartinhaler Tracker, Nexus6 Limited, Auckland, New Zealand), which recorded the date and time (to the nearest second) each time an inhaler was actuated. These monitors were 99·7% accurate in recording actuations in bench testing19 and were used in accordance with strict trial quality control processes.20 Participants were not aware of the exact capabilities of the monitors. ABSTRACT.DEFINITIONS OF HIGH, MARKED AND EXTREME BETA-AGONIST USE EPISODES: ABSTRACT.HIGH USE: For SMART, this was defined as >8 actuations of budesonide/formoterol in excess of the four maintenance doses per 24-h period i.e., equivalent to >12 actuations in total. For Standard, this was defined as >16 actuations of salbutamol per 24-h period. These thresholds were based on the dose limits of beta-agonist use requiring medical review, defined by the action plans implemented in this study,21, 22 and supported by the results of the short-term bronchodilator equivalence of formoterol 6 µg to salbutamol 200 µg, with repeat dosing in acute asthma.23, 24 In accordance with their action plans, participants were advised to seek medical review at these high use thresholds. ABSTRACT.MARKED USE: For SMART, this was defined as >12 actuations of budesonide/formoterol in excess of the four maintenance doses per 24-h period i.e., equivalent to >16 actuations in total. For Standard, this was defined as >24 actuations of salbutamol per 24-h period. These thresholds were based on 1.5 times the limits of beta-agonist use requiring medical review, defined by the action plans.21, 22 ABSTRACT.EXTREME USE: For SMART, this was defined as >16 actuations of budesonide/formoterol in excess of the four maintenance doses per 24-h period i.e., equivalent to >20 actuations in total. For Standard, this was defined as >32 actuations of salbutamol per 24-h period. These thresholds were based on twice the limits of beta-agonist use requiring medical review, defined by the action plans.21, 22 ABSTRACT.HIGH, MARKED, AND EXTREME BETA-AGONIST USE WITHOUT MEDICAL REVIEW: The number of days of high, marked, or extreme beta-agonist use without medical review within 48 h was determined using the following rules: for every high, marked, or extreme beta-agonist use day, the ‘index day’, the database was checked to determine if the patient attended for medical review, which could include primary care clinic, an after-hours clinic or hospital attendance, either on the day of overuse or the next day. This 48-h window was defined as per the Standard action plan, which specifies that the patient should attend for medical review ‘within 1 to 2 days’ in the setting of worsening asthma recognised by symptoms including ‘reliever only lasting 2–3 h’.22 The SMART plan advises patients to seek medical review on the same day if more than 12 actuations of budesonide/formoterol are taken.21 If the participant attended for medical review, then overuse occurring on the index day, the day of medical review and in the 7 days after medical review was not counted as overuse without medical review, as a ‘stand-down’ period. In effect, overuse occurring on these days was ‘permissible’ as the patient had attended for medical review in the setting of this exacerbation. This criterion was chosen as the american thoracic society (ATS)/european respiratory society (ERS) definition of severe exacerbations separate exacerbations by 7 days.25 If a participant attended for repeated medical reviews during the stand-down period, then the 7-day period where overuse without medical review is not counted, was restarted. This approach was used as the object of this analysis was to explore the relationship between the ‘index’ overuse episode and the first episode of medical review following this. ABSTRACT.OUTCOMES: In this exploratory analysis, no particular primary outcome was specified and there is no adjustment for multiple comparisons. The outcome variables were firstly the proportion of participants in each arm of the RCT with at least one episode of overuse for each type of beta-agonist overuse; high, marked and extreme. A second outcome was the number of days of overuse for each type of beta-agonist overuse. A third outcome was the number of days of overuse without medical review within 48 h for each type of beta-agonist overuse. The final outcome was the proportion of days of overuse without medical review within 48 h compared to the total number of days of overuse for each type of beta-agonist overuse. Medication use in the 14 days prior to and following a severe exacerbation was also examined. A severe exacerbation was defined according to ATS/ERS criteria25 as: (a) the use of systemic corticosteroids for at least 3 days; or (b) a hospitalisation or Emergency Department visit because of asthma, requiring systemic corticosteroids. Courses of corticosteroids separated by 7 days or more were treated as separate severe exacerbations. The date of the first day of corticosteroid therapy was Day 0. Medication use for the 14-day period before and up to 14 days after Day 0 was extracted from the data set for each participant with at least one severe exacerbation. For patients with repeat exacerbations, a minimum interval of 28 days between exacerbations was required to avoid overlap of data within the same participants. ABSTRACT.STATISTICAL METHODS: The relative risk of at least one episode of overuse by overuse type was by simple contingency table analysis and expressed as a relative risk. The relative rates of days of high, marked and extreme beta-agonist overuse was by Poisson regression with an offset for the adjusted days of treatment exposure, and a further adjustment for over-dispersion. SAS version 9·4 was used. ABSTRACT.DATA AVAILABILITY STATEMENT: Data is available on application to the corresponding author ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary Information

TITLE: Use of a self-rating scale to monitor depression severity in recurrent GP consultations in primary care – does it really make a difference? A randomised controlled study ABSTRACT.BACKGROUND: Little information is available about whether the use of self-assessment instruments in primary care affects depression course and outcome. The purpose was to evaluate whether using a depression self-rating scale in recurrent person-centred GP consultations affected depression severity, quality of life, medication use, and sick leave frequency. ABSTRACT.METHODS: Patients in the intervention group met their GP regularly at least 4 times during the 3 months intervention. In addition to treatment as usual (TAU), patients completed a self-assessment instrument (Montgomery-Asberg Depression Rating Scale) on each occasion, and then GPs used the completed instrument as the basis for a person-centred discussion of changes in depression symptoms. The control group received TAU. Frequency of visits in the TAU arm was the result of the GPs’ and patients’ joint assessments of care need in each case. Depression severity was measured with Beck Depression Inventory-II (BDI-II), quality of life with EQ-5D, and psychological well-being with the General Health Questionnaire-12 (GHQ-12). Data on sick leave, antidepressant and sedatives use, and care contacts were collected from electronic patient records. All variables were measured at baseline and 3, 6, and 12 months. Mean intra-individual changes were compared between the intervention and TAU group. ABSTRACT.RESULTS: There were no significant differences between the intervention and control group in depression severity reduction or remission rate, change in quality of life, psychological well-being, sedative prescriptions, or sick leave during the whole 12-month follow-up. However, significantly more patients in the intervention group continued antidepressants until the 6 month follow-up (86/125 vs 78/133, p < 0.05). ABSTRACT.CONCLUSIONS: When GPs used a depression self-rating scale in recurrent consultations, patients more often continued antidepressant medication according to guidelines, compared to TAU patients. However, reduction of depressive symptoms, remission rate, quality of life, psychological well-being, sedative use, sick leave, and health care use 4-12 months was not significantly different from the TAU group. These findings suggest that frequent use of depression rating scales in person-centred primary care consultations has no further additional effect on patients’ depression or well-being, sick leave, or health care use. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01402206. Registered June 27 2011(retrospectively registered). ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12875-016-0578-9) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Depression is a leading cause of disability and affects 10 to 15% of the population of most countries in the world [1, 2]. A majority of patients with depression are treated in primary care, and approximately 75% of antidepressants are prescribed by general practitioners (GPs) [3, 4]. The usual treatment for depression in Swedish primary care, as in primary care in other countries, depends upon symptom severity. Most people treated for depression in primary care receive one or more of the following: early reassessment (watchful waiting) and symptom monitoring by their GP, cognitive behavioural therapy or other psychotherapy, and antidepressant medication [5, 6]. Depression guidelines recommend that GPs regularly use self-rating scales to evaluate and monitor symptoms in patients with depression [5, 6]. These guidelines, however, have been based largely on clinical expertise rather than on the results of randomised controlled trials [3]. Furthermore, most self-rating scales recommended for detecting and monitoring depression were designed in psychiatric care settings and have not been adapted for use in primary care [3]. Little information is thus available about whether the use of such instruments in primary care affects depression symptoms, treatment, rehabilitation, or recovery [7]. We know that using such scales for screening does not improve detection or management of depression in settings such as primary care [8]. However, we do not know whether using a self-rating scale to follow the course of depression in person-centred primary care consultations improves treatment and outcomes of depression. The depression rating scale most commonly used in Swedish primary health care is the Montgomery-Asberg Depression Rating Scale [9]. However, its use has mainly been evaluated in psychiatric settings [9, 10]. MADRS-S is sensitive to changes in symptoms of depression and is therefore a valuable tool in person-centred consultations [10–12]. However, a qualitative study based on interviews with Swedish GPs showed that many GPs thought that MADRS-S was of limited utility in consultations [13]. It is thus still unclear whether or not future guidelines should recommend mandatory use of depression self-rating scales in primary health care. The aim of the current study was to evaluate in a randomised control trial whether using a depression rating scale (MADRS-S) in recurrent person-centred GP consultations affected depression severity, quality of life, overall psychological well-being, antidepressant and sedative use, sick leave, and health care use in a long time perspective. BODY.METHODS: The PRI-SMA study (PRImary care Self-assessment Montgomery-Asberg) was a multicentre, controlled trial that took place at primary health care centres (PHCCs) and was randomised at the GP level. The GPs randomised to the intervention used a patient self-rating scale at recurrent monthly patient visits in addition to providing usual care. The GPs randomised to the control condition provided treatment as usual (TAU). The intervention lasted for 3 months. Adult patients who presented with a recent depressive episode (mild/moderate depression) at the PHCC were recruited to the study by the GPs. The trial took place at 22 Swedish PHCCs between March 2010 and December 2013. All 98 PHCCs in the region were invited to participate in the intervention; 22 agreed to participate. BODY.RANDOMISATION OF GPS AT PRIMARY HEALTH CARE CENTRES: All GPs took part in an information meeting about the study. All GPs also met with the study leaders when the leaders visited each participating PHCC at the time of the intervention start-up at that PHCC. Before the intervention started, the GPs at each PHCC were randomised to either intervention treatment or TAU. All GP names were written on slips of paper and mixed in a container, and an administrative employee blinded to the aim of the trial drew names. The GP whose name was first drawn was assigned to the intervention group, the GP whose name was drawn second was assigned to the control group, and so on until all names were drawn. BODY.STUDY PROCEDURE: Patient recruitment took place at one PHCC at a time. During the 2 week recruitment period, a research nurse worked full-time at the PHCC to facilitate patient recruitment, collect patient data, and support PHCC staff. BODY.INCLUSION OF PATIENTS: Study participants were patients aged 18 and up who visited the PHCCs and were identified and diagnosed by a GP with a new episode of mild/moderate depressive disorder [14]. During the 2 week recruitment period, all randomised GPs who met patients they suspected of having depression used the PRIME-MD diagnostic instrument to confirm or reject the diagnosis of depression in accordance with DSM-IV criteria [14]. All patients who fulfilled the diagnostic criteria for depressive disorder, i.e. mild to moderate (BDI score ≤36), were asked if they would like to participate in the study. Inclusion criteria were: written informed consent, age ≥18 years, diagnosed with mild to moderate depressive disorder and either not prescribed antidepressant medication or had no changes in antidepressant medication during the preceding 2 months. Exclusion criteria were: lack of written informed consent, antidepressant medication introduced or changed during the 2 months prior to baseline, diagnosed with severe depressive disorder (BDI-II >36, confirmed by diagnostic procedure by GP), diagnosed with severe mental disorder (i.e., bipolar disorder, antisocial personality disorder, psychosis, substance use disorder, or other serious mental disorder), suicidal ideation or earlier suicide attempts, did not speak or understand Swedish, and/or had cognitive disabilities that made it difficult or impossible to complete the assessment instruments, including MADRS-S. BODY.INTERVENTION: The intervention consisted of using a patient depression self-rating scale (MADRS-S) in recurrent monthly consultations during the 3-month intervention. Patients made 4 visits to their GPs, at which time they completed MADRS-S to monitor changes in their depressive symptoms that were then discussed in the person-centred consultation [15, 16]. MADRS-S was used as a supplement to, rather than as a substitute for, TAU. The GPs randomised to the group that provided the intervention received four hours of guidance about how to include the results of MADRS-S in the person-centred consultation. The intervention GPs also received a video CD with the same pre-recorded information. The person-centred consultations involved patients and GPs collaborating to increase patients’ ability to manage their depression. The guidance therefore included a reminder to the GPs that MADRS-S was used for the sake of the patients rather than the GPs. BODY.TREATMENT AS USUAL: The GPs randomised to the group that would provide TAU were instructed to manage patients with depression the same way they usually did (but with the addition of the diagnostic procedure in the initial consultation). In general Swedish GPs are very knowledgeable about and use of person-centred consultation methods in their daily practice of the kind described in Maguire 2002 [16]. Three months after baseline, patients in the control group were followed up in an appointment with a nurse at the PHCC. BODY.DATA COLLECTION PROCEDURE: A study nurse collected data from participants in the intervention and control groups during the first visit (baseline), at a follow-up visit to the PHCC at the end of the intervention (3 months after baseline), and by postal questionnaires 6 and 12 months after baseline (see Additional files 1 and 2). BODY.MEASURES: BODY.BACKGROUND VARIABLES: Background information was obtained by a questionnaire at the patients’ first visit to the PHCC. Age was measured as age in years, gender as female or male. Marital status was dichotomised as single or married/cohabiting. Children living at home was dichotomised as participants who had children <18 years living at home vs. participants not having children living at home. Educational level was identified as as lower educational level (primary school or vocational school), middle educational level (high school) and higher educational level (college or university). Employment status was dichotomised as working/studying vs. unemployed/retired. Place of birth was dichotomised as born in one of the Nordic countries vs. born outside the Nordic countries. Smoking status was categorised as “non-smokers” (participants who had not smoked during the last year), “sometimes smokers” (those who only smoked a single cigarette per week or more seldom), and “smokers” (more frequent smoking). Leisure-time physical activity was categorised as i) no or almost no physical activity during leisure time, ii) activity ≥4 hrs. per week, and iii) intensive (several times a week and vigorously). BODY.OUTCOMES: Depression severity; Assessed before (at baseline) and after the intervention with the Beck Depression Inventory-II (BDI-II) and categorised as mild (BDI-II score >13-19), moderate (BDI-II score 20-28), and high moderate (BDI-II score, 29-36) [17, 18]. Remission; Identified as a BDI-II score ≤ 13 at 3-month follow up. Quality of life; Measured with the standardised EQ-5D instrument [19, 20]. Overall psychological well-being; Assessed with the 12-item General Health Questionnaire (GHQ-12) (response scale 0-3) [21]. Prescriptions for antidepressants; Information on this measure was obtained from patients’ electronic patient records (EPRs) and from responses to the patient questionnaires and dichotomised as yes (used) or no (did not use). Guidelines indicate that treatment should not be stopped before 6-12 months after recovery [5]. Prescriptions for sedatives; Information on prescriptions for sedatives was obtained from patients’ electronic patient records (EPRs) and from responses to the patient questionnaires and dichotomised as yes (used) or no (did not use). Sick leave; Information on sick leave was obtained from EPRs and responses to the patient questionnaires. Sick leave was used both as a dichotomous variable (yes or no) and as number of days of sick leave. Health care use; This measure comprised information on number of visits to GPs, number of visits to nurses, number of visits to psychologists/psychotherapists, and total number of visits to the PHCC. Information was obtained from EPRs by study personnel and divided into the number of visits between 0 and 3 months, and 4 and 12 months. BODY.STATISTICAL METHODS: The student’s t-test (paired) was used to compare mean intra-individual changes in the intervention and TAU groups. Two-sided Chi-square or Mann Whitney U-tests were used to compare frequencies. Statistical significance was set at p < 0.05. Logistic regression models were used to examine whether changes in the BDI-II, EQ-5D, and GHQ-12 variables from baseline to 3, 6 and 12 months follow-ups were associated with sex, age, education, antidepressant medication, and group assignment (intervention/TAU).The results are presented as odds ratios (OR) with 95% confidence intervals (CI). All analyses were performed in SPSS. All analyses were conducted on an intention to treat basis. BODY.POWER CALCULATION: Power was calculated for the BDI-II-score outcome on the basis of empirical experience of the variation in this factor, taking into account that GPs were the unit of randomisation. The power estimate was based on the assumption of an alpha error = 0.05 and a beta error = 0.20 (power = 0.80). Given an expected effect difference of 2 units (BDI-II) over time between the intervention and TAU groups, we calculated that we would need 105 patients in each group to achieve sufficient statistical power. To safeguard for dropouts, we recruited 20% more patients than the power calculation indicated that we needed. BODY.ETHICS: The Regional Ethical Review Board in Gothenburg, Sweden approved this study (Dnr 746-09; T612-10). Prior to inclusion and after receiving oral and written information about the study, participants provided written informed consent. The trial was registered at ClinicalTrials.gov (Identifier: NCT01402206) [22]. BODY.RESULTS: Of the 91 participating GPs, 45 were randomised into a group that provided the intervention and 46 into a group that provided TAU. The participating GPs enrolled 258 patients, and the flow of the patients through the study is shown in Fig. 1 [23]. There was no significant difference in participation rate in the patients in the intervention and control groups at the 3-, 6- or 12-months follow-ups.Fig. 1PRI-SMA-Study (CONSORT Flowchart) Table 1 presents the background characteristics of the study population. No significant differences were found between the participants in the intervention and TAU groups at baseline.Table 1Baseline characteristics of participants in the PRI-SMA trial (n = 258) CharacteristicsTotal n (%)Intervention n (%)TAU¤ n (%) p Participants 258 125 133 Age (years, mean ) 43.48 44.84 42.19 0.2  men 76 (29.5) 31 (24.8) 45 (33.8)  women 182 (70.5) 94 (75.2) 88 (66.2) 0.1 Marital status  single 118 (45.7) 61 (48.8) 57 (42.9)  married/cohabiting 140 (54.3) 64 (51.2) 76 (57.1)  children <18 years at home 82 (43.2) 36 (40.4) 46 (45.5) 0.5 Lower educational level 34 (13.2) 17 (13.7) 17 (12.8) Middle educational level 115 (44.7) 61 (49.2) 54 (40.6) High educational level 108 (42.0) 46 (37.1) 62 (46.6) 0.3 Employment  working/studying 181 (80.8) 90 (81.1) 91 (80.5)  unemployed/retired 43 (19.2) 21 (18.9) 22 (19.5) 0.9 Born outside the Nordic countries 41 (16) 19 (15.3) 22 (16.7) 0.8 Smoking (yes or sometimes) 75 (29.4) 36 (29.3) 39 (29.5) 0.9 Leisure-time physical activity  never 109 (42.4) 57 (46) 52 (39.1)  at least 4 hrs/week 124 (48.2) 55 (44.4) 69 (51.9)  intensive 24 (9.3) 12 (9.7) 12 (9,0) 0.5 Depression  mild (BDI-II 12-19) 32 (13) 16 (13.6) 16 (12.4)  moderate (BDI-II 20-28) 82 (33.2) 39 (33.1) 43 (33.3)  high moderate (BDI-II 29-36) 68 (27.5) 35 (29.7) 33 (25.6) 0.9 ¤ TAU treatment as usual There was a statistically significant difference between participants and drop-outs during the study concerning age (mean age 44.3 in participants, mean age 37.3 in drop-outs, p = 0.02), gender (male 14/62, 22.6%, female 16/166, 9.6%, p = 0.034), and ethnicity (born in Sweden 21/194, 10.8%, and born outside Sweden 9/32, 28.1%, p = 0.035) Figures 2, 3, and 4 shows depression symptoms (BDI-II), quality of life (EQ-5D), and overall psychological well-being (GHQ-12) in intervention and TAU groups at baseline and 3, 6, and 12 months (boxplots). All patients who participated in the study, both those in the intervention and those in the TAU group, improved in all three variables between baseline and 3 months but there were no significant differences in mean changes (mean of intra-individual ∆) between the groups. Improvements were substantial in both groups; at the 3-month follow-up, nearly half the patients no longer had depression (BDI <13) (49% in the intervention and 47.3% in the TAU group, p = 0.89). We also performed logistic regression analyses. The models showed that at the 12-month follow-up, older age was associated with improved BDI-II score (OR 1.05, CI 1.01-1.99), and a higher level of education was associated with improved EQ-5D score (OR 3.9, CI 1.77-8.73). The factors added to the model (sex, age, education, antidepressant medication, and participation in the intervention or TAU group) explained the same amount of variation in both the intervention and TAU groups. Thus, the difference between the groups remained non-significant (data not shown). No adverse events were reported from intervention or TAU group.Fig. 2Depression severity (BDI-II values) in the intervention and TAU groups. Boxplots for baseline, 3-months follow up, 6-months follow up, and 12-months follow up. The outcome variable is presented as box plot with medians, minimum and maximum values and lower and upper quartiles. Y-axis: BDI-II, X-axis: time 0, 3, 6, 12 months. Outliers (circle) are cases with values between 1.5 and 3 times the interquartile range (5 cases with BDI-II ≥50 in patients with difficulties in Swedish language, where complementary diagnostic procedure by the GP ended up on medium depressive disorder) Fig. 3Quality of life (EQ-5D values) in the intervention and TAU groups. Boxplots for baseline, 3-months follow up, 6-months follow up, and 12-months follow up. The outcome variable is presented as box plot with medians, minimum and maximum values and lower and upper quartiles. Y-axis: EQ-5D, X-axis: time 0, 3, 6, 12 months. Outliers (circle) are cases with values between 1.5 and 3 times the interquartile range, extremes (star) are cases with values more than 3 times the interquartile range Fig. 4circle) are cases with values between 1.5 and 3 times the interquartile range Table 2 shows differences between the intervention and TAU groups in the percent of patients who had prescriptions for antidepressants and sedatives. At baseline, 22% of patients in the intervention group and 32% of patients in the TAU group were on maintenance antidepressant medication. After 3 months, the proportion of patients with prescriptions for antidepressant medication was 72% in both the intervention and the TAU group. At the 6-month follow-up, the proportion of patients taking antidepressants had dropped to 69% in the intervention group and 59% in the TAU group (p = 0.007). At the 12-month follow-up, there were no significant differences in the percent of patients who had prescriptions for antidepressants or sedatives in the intervention and TAU groups (Table 2).Table 2Number and percent of patients who had prescriptions for antidepressants and sedatives and stated medication at baseline and 3-, 6-, and 12-months follow up, respectively, in the intervention and TAU groups Baseline3 months follow-up6 months follow-up12 months follow-up n (%) n (%) n (% ) n (%)Antidepressants  Intervention (n = 125) 27 (22) 90 (72) 86 (69)** 74 (59)  TAU (n = 133) 43 (32) 96 (72) 78 (59) 77 (58) Sedatives  Intervention 26 (21) 64 (51) 53 (42) 52 (42)  TAU¤ 32 (24) 73 (55) 56 (42) 51 (38) ¤ TAU treatment as usual **Significantly higher proportion of patients in intervention group still on antidepressants after 6 months, p = 0.007 Table 3 shows the mean number of days of sick leave by patient group. There were no significant differences in the percentage of patients on sick leave in the intervention and TAU groups between baseline and 3 months, or 4 -12 months, or during the entire study period. The mean total duration of sick leave (days) was not significantly different between the intervention and TAU groups (Table 3).Table 3Number and percent of individuals on sick leave, mean days of sick leave, and p-values for difference in mean days of sick leave between participants in intervention and TAU groups, based on information obtained from electronic patient records InterventionTAU n (%)Mean days of sick leaveSD n (%)Mean days of sick leaveSD P for difference mean days0-3 months 31(25) 63.1 29.8 48 (36) 55.8 27.7 0.221 4-12 months 35(28) 100.8 87.3 47(35) 102.7 85 0.922 Total 0-12 months 49(39) 124.8 102.5 64 (48) 123.3 85.0 0.942 BODY.HEALTH CARE USE: Mean number of visits at the PHCC, including visits to GP, nurse, psychologist/therapist and total visits to PHCC, was compared between intervention and TAU groups. All information was obtained from EPRs (Table 4). We also compared the total number of visits. There were no significant differences between the intervention and TAU groups regarding these outcomes, during the total 12-month follow-up period. However, during the 0-3 month period, the TAU group made significantly more visits to psychologists/psychotherapists than the intervention group and the intervention group made significantly more total visits to the PHCC than the TAU group.Table 4Number of patients’ contacts with GPs, nurses, and psychologists between baseline to 3 months, and 4 to12 months for intervention and TAU groups, based on information obtained from electronic patient records Profession Type of contact Intervention 0-3 months m (SD) TAU 0-3 months m (SD) p  GP  visit  3.44 (1.214)  2.59 (1.354) 0.066  Nurse  visit  0.32 (0.829)  0.32 (0.875) 0.878  Psychologist/Therapist  visit  0.40 (1.320)  0.89 (1.776) 0. 0001  Total visits to PHCC  visit  4.16 (2.398)  3.81 (2.692) 0.006 Intervention 4-12 months m (SD) TAU 4-12 months m (SD) p  GP  visit  2. 54 (2.337)  2.38 (2.595) 0.301  Nurse  visit  0. 68 (1.484)  0.62 (1.551) 0.581  Psychologist/Therapist  visit  0. 90 (2.504)  0.83 (1.908) 0.401  Total visits to PHCC  visit  4. 11 (4.586)  3.83 (4.403) 0.619 Intervention total 0-12 months m (SD) TAU total 0-12 months m (SD) p  GP  visit  8.26 (5.842)  7.64 (5.976) 0.304  Nurse  visit  0.99 (1.978)  0.94 (2.088) 0.967  Psychologist/Therapist  visit  1.30 (3.129)  1.73 (3.222) 0.167  Total visits to PHCC  visit  8.26 (5.842)  7.64 (5.976) 0.812 Bold numeral indicates statistically significant p-value BODY.DISCUSSION: In this RCT, which evaluated use of a depression self-rating scale in recurrent person-centred GP consultations, more patients in the intervention than control group adhered to antidepressant treatment for the recommended 6 months. However, compared to TAU, there were no significant differences between the effects on depression symptom reduction, quality of life or overall psychological well-being, sedative use, sick leave, or health care use. These findings suggest that recurrent use of depression self-assessment scales in person-centred primary care consultations does have an effect on antidepressant medication adherence, but has no further additional effects, compared to the treatment usually provided in Swedish primary care. BODY.STRENGTHS AND WEAKNESSES OF THE STUDY: The intervention was conducted in ordinary primary care rather than advertising for participants, which would have resulted in a sample that was not representative of patients in primary health care. The study compared the intervention with a TAU group rather than using a control group that consisted of patients on a waiting list. Studies of the effects of cognitive behavioural therapy show that being on a waiting list has a nocebo effect (i.e. results in worsening depression symptoms), which in turn makes intervention effects appear larger than they really are [24, 25]. We had access to all patients’ EPRs, a good source of information on prescriptions, sick leave, and health care use. In the current study, we had the opportunity to observe the course of depression symptoms, perceived quality of life, sick leave, and health care use for a full year after baseline. Another major strength is that a research nurse was stationed at each PHCC during the recruitment period. The nurse collected baseline data, gathered oral and written consent, and supported the staff at the site, functions that primary care personnel stress are important to make it easier to combine clinical work and research [26].We were well aware of the possible obstacles to conducting a RCT at a PHCC as described by Richter and Roy-Byrne [27, 28]. We randomised at the GP level. Randomisation at the patient level would have necessitated changing doctor for some patients, or GPs trained in the intervention would have provided the intervention to some patients but treatment as usual to others, increasing the possibility of contamination. We chose not to randomise at the PHCC level, as regional and socioeconomic differences between areas served by the PHCCs might have affected the results. Another limitation was absence of a blinded control arm. The control GPs were aware that their colleagues at the same PHCC were providing the intervention, and this might have affected treatment as usual, even if they were requested to continue patient care as usual. This possible bias would support the notion of randomisation at PHCC level, but this would in turn increase the risk of e.g. selection effects. Another limitation is that the intervention of recurrent use of self-rating scales also included regular GP visits, and it is not possible to separate possible effects of the regular GP visits from effects of the patient’s frequent self-rating monitoring. However, during the 3-month intervention, patients in the TAU arm made only 0.85 fewer visits to their GPs than patients in the intervention arm. The frequency of visits in the TAU arm was the result of the GPs’ and patients’ joint assessments of care needs in each case. That frequency of patient visits did not differ markedly in the TAU arm is likely attributable to the current wide-spread emphasis on person-centred care in Swedish primary care. All patients included had a new episode of mild/moderate depression, but the selection procedure did not differentiate between recurrent or first episode patients. There is some evidence that recurrent depression has slower remission [29], but according to the similar frequency of maintenance antidepressant medication in both intervention and TAU groups at inclusion, we can assume that the rate of recurrent depression disorder was the same in both arms. Further, the high number of outcomes, assessed at baseline and at 3, 6, and 12 months, necessitated multiple statistical comparisons, thus increasing the possibility of type I errors. Although the low p-values indicated that the few significant findings were not likely due to chance, the results need further confirmation in other studies. A limitation is also that we did not use more advanced statistical models to account for the correlation between patients treated by the same GP and to handle all observations of the patient instead of pairwise comparisons (e.g. mixed models). BODY.DROP OUTS: Among the study drop outs, 37 individuals belonged to the intervention group and 48 to the TAU group. At the 3 and 6 month follow-up, participation rate was somewhat lower in the control group, but at the 12 month follow up, participation rate was almost the same in intervention and control groups; 70% and 69%, respectively (Fig. 1). BODY.GENERALISABILITY: The study population represented patients treated by GPs for mild and moderate depression in primary health care. It included patients from health care centres in urban and rural areas and areas characterised by differing socioeconomic status. Health care is heavily subsidised in Sweden; patients pay a small per-visit fee and their yearly total co-payment is capped at approximately £100. The study results can be generalised to other countries with similar health care systems. The RCT had a representative selection among the Swedish speaking population. One could argue that the exclusion of non-speaking Swedish patients may have limited the study’s generalisability. Unfortunately, it was not possible to translate and validate the instruments or to use an interpreter at every contact with study staff. In this study, we chose to use MADR-S as the basis for patient education and collaborative care. These two factors can facilitate better depression outcomes [29–31]. Depression is complex, and the outcome of treatment often depends on the GP’s ability to connect with the patient during the consultation and her/his ability to choose proper treatment in agreement with the patient. Use of self-rating scales may function as one piece of the depression treatment puzzle, but as in many difficult jigsaw puzzles, more than one piece is needed to complete the picture. Thus, the Swedish National Board of Health and Welfare, i.e. the body that establishes the national guidelines for depression treatment, has expressed interest in whether it is meaningful to routinely use self-rating scales for patients with mild to moderate depression in primary care. We chose to use MADRS-S because it is the most commonly used depression self-rating scale in Sweden. About one third of all GPs state that they sometimes use MADRS-S, although not regularly [13, 32]. MADRS-S is designed to be sensitive to change and is often used in RCT studies that evaluate antidepressant treatment [12]. The study shows that use of self-rating scales during regular visits facilitates adherence to pharmacological treatment according to guidelines, in that once medication with antidepressants has been initiated, this treatment should be continued for at least 6 months [5]. On the other hand, the study also shows that treatment as usual, as it is practiced in the Swedish primary care of today, in most cases not including the use of self-rating scales, is just as efficient concerning reduction of depressive symptoms, perceived quality of life and general health, as well as sick leave frequency and duration, which is supported by the results from several international studies [33]. BODY.STRENGTHS AND WEAKNESSES IN RELATION TO OTHER STUDIES: To our knowledge there have been few or no studies that have evaluated the effect of recurrent use of self-rating scales regarding depression in relation to the above described outcomes. Use of antidepressants for the period mandated by current guidelines was associated with neither an improvement nor a worsening of patients’ symptoms during the one-year study period. However, using antidepressants for the length of time called for in guidelines has been shown to reduce risk of relapse [34]. BODY.CLINICAL IMPORTANCE OF THE STUDY: More discussion is needed as to how self-rating scales might be best implemented in primary care, how to manage depressed patients using these instruments, and how to integrate this into the consultation. The feasibility of meaningful and person-centred use of self-rating scales, continuity, and accessibility in the care of mild to moderate depression in the clinical setting is of great interest. Engaging the patient with a cognitive and person-centred approach and with regular consultations and follow-ups is a way to empower the patient, give the patient knowledge and tools to handle depression, follow his or her illness course, and create a basis for regaining of function [2, 6, 30]. Continuity and close regular follow-ups facilitate care that is adjusted to the individual's explicit needs and requirements and is an absolute condition for person-centred care [35]. Requirements for treatment are also reached in cooperation and collaboration with the patient, facilitating understanding and communication [36]. The continuous use of self-rating scales in the treatment of primary care patients with depression might support this process of care. BODY.CONCLUSIONS: When GPs used a depression self-rating scale in recurrent consultations, patients more often continued antidepressant medication according to guidelines, compared to TAU patients. However, concerning outcomes in terms of depression symptoms, quality of life, overall psychological well-being, consumption of care, and sick leave the use of self-rating scales did not seem to increase treatment effects compared to the usual treatment provided in primary care. Thus, the use of depression self-rating scales should perhaps not be mandatory in primary health care but rather left to the discretion of the GP and the patient.

TITLE: Comparison of Lactobacillus Sporogenes plus mineral oil and mineral oil alone in the treatment of childhood functional constipation ABSTRACT.BACKGROUND:: Functional constipation is one of the most prevalent childhood gastrointestinal disorders. We evaluated effects of adding a probiotic to mineral oil in the treatment of functional constipation in children. ABSTRACT.MATERIALS AND METHODS:: This controlled trial was conducted on 60 children (2 to 14 years old) with functional constipation (Rome III criteria). Children were allocated to receive the synbiotic (Lactol®, composed of Lactobacillus Sporogenes, 1 Tab/20 kg/d) plus mineral oil (Paraffin 1 ml/kg/d) or the mineral oil alone for two months. Symptoms of constipation including defecation frequency, stool form, strain and pain at defecation, incomplete evacuation and soiling were assessed and compared before and after the intervention. After the treatment period, the two groups were also compared with regards to subjective global assessment of improvement. ABSTRACT.RESULTS:: After the treatment, stool frequency increased in both groups (P < 0.001), with greater increase in synbiotic + mineral oil group (P = 0.001). Frequency of hard/very hard stool and frequency of painful defecation decreased similarly in both groups (P < 0.001). Straining at defecation, incomplete evacuation, and soiling decreased in both groups (P < 0.001), but more decrease was seen in the synbiotic + mineral oil group (P < 0.05). Finally, there was a better global improvement in the synbiotic + mineral oil group (P < 0.05). No severe side-effects were observed in any group. ABSTRACT.CONCLUSION:: Adding the synbiotic Lactol® (containing Lactobacillus Sporogenes) to mineral oil can increase the improvement in the constipation symptoms of children without specific side-effects. BODY.INTRODUCTION: Constipation is one of the most prevalent gastrointestinal disorders among children with the reported prevalence varying from about 1-8% in the pediatric population. It accounts for 3-5% of visits to pediatricians and up to 25% of visits to pediatric gastroenterologists.[1] Disease burden and estimated healthcare costs are considerable and it can significantly impair quality of life of patients in both mental and physical health aspects.[23] In most of the cases (90-95%), no organic cause is found for constipation leading to the diagnosis of functional constipation in the child.[4] Thus, treatment strategy mainly involves educating the family and dietary modification. Disimpaction and maintenance oral/rectal medications are administered when needed. Common current medications for maintenance therapy include mineral oil, lactulose, milk of magnesia, polyethylene glycol powder, and sorbitol.[56] Because the etiology of the disease is not clearly understood, there is no specific treatment for functional constipation, and despite various treatments, only about half of the children with functional constipation demonstrate long-term improvement.[5] There is growing interest about the efficacy of probiotics in the treatment of functional as well as organic gastrointestinal disorders. Probiotics have been reported to be effective in the treatment of various disorders including inflammatory bowel disease, irritable bowel syndrome, travelers’ diarrhea, and constipation. Because colonic microflora can affect the motility of the colon, an imbalance in the colonic microflora is proposed to have a role in the pathophysiology of chronic constipation. Also, some kind of probiotics, such as bifidobacteria and lactobacilli, by producing acids (lactic, acetic), result in lowering the pH of the colon, which in turn can enhance peristalsis of the colon and decrease colonic transit time.[78] Several reports are available on the beneficial effects of probiotics on symptoms of constipation, mainly in adults.[8] Studies showed that probiotic strains, such as B. Infantis, increase defecation frequency and soften stools. However, limited information is available on the efficacy and safety of probiotics in the treatment of children with constipation and available studies have had controversial results.[8] We evaluated the effects of adding a synbiotic containing Lactobacillus Sporogenes strain to mineral oil in the treatment of functional constipation in children. BODY.MATERIALS AND METHODS: BODY.PATIENTS AND SETTING: This controlled clinical trial was conducted on children referring with complaint of constipation to an outpatient clinic of pediatric gastroenterology, at the Alzahra University Hospital, Isfahan city (IRAN), from 2010 to 2011. Children 2 to 14 years of age diagnosed to have functional constipation based on the Rome III criteria were included consecutively.[9] Those with immunocompromised condition or other severe diseases, and those receiving antibiotic in the previous four weeks were not included.[10] Exclusion criteria included experiencing severe side-effects, gastroenteritis, or receiving antibiotic for any reason during the study period. Considering alpha = 5%, study power = 80%, and expecting at least one score change in stool consistency scale, the sample size was calculated as 25 subjects in each group. The study was approved by the ethics committee of the Azad University, Najaf Abad Branch, and informed consent was obtained from all children's parents. Also, the study was registered in the Iranian Registry of Clinical Trials (IRCT201207311579N1). BODY.INTERVENTION: Children were alternately allocated to the synbiotic and control groups. In the synbiotic group, subjects received synbiotic (Lactol®, Bioplus Life Sciences Pvt. 1 Tab/20 kg/d) plus mineral oil (Paraffin 1 ml/kg/d) for eight weeks. The synbiotic used in this study was composed of the following; Lactobacillus Sporogenes (15 × 107 Spores), Fructo-Oligosaccharides, Microcrystalline Cellulose, Sodium starch Glycolate, Povidone, Hypermellose Stearate, Sillicon Dioxide, and Propylene Glycol. Subjects in the control group received mineral oil (Paraffin 1 ml/kg/d) for eight weeks. Toilet training and dietary advice were equally administered for both groups.[11] BODY.ASSESSMENTS: Diagnosis of functional constipation was made according to the Rome III criteria by a pediatric gastroenterology specialist. The criteria were two or more of the following: 1) Two or fewer stools per week, 2) hard or very hard stools or painful stool, 3) Passage of very large stool, 4) Stool retention “once a week” or more often, 5) History of large fecal mass in rectum, and 6) Soiling “once a week” or more often. Those who fulfiled the criteria for irritable bowel syndrome were not included into the study.[9] A trained general physician interviewed with each subject, and a questionnaire including demographic data and symptoms of constipation, based on the Questionnaire on Pediatric Gastrointestinal Symptoms-Rome III Version, was completed.[12] Major symptoms included defecation frequency (1 = two times a week or less to 5 = more than three times a day), stool consistency (1 = very hard to 5 = watery), stool retention (yes/no), and painful defecation (yes/no). Other symptoms included urgency, straining, passing mucus, and feeling of incomplete evacuation which were scored from 0 (never) to 4 (always) and soiling which was scored from 0 (never) to 5 (every day). Subjects were interviewed at the end of the intervention (8th week) with the same questionnaire including compliance and side-effects as well, and a subjective global improvement (SGI) scale which was scored from 0 (significantly worsen) to 7 (completely improved).[13] BODY.STATISTICAL ANALYSES: Data were analyzed using SPSS software for windows Version 16.0. Statistical tests included independent sample t-test for comparison of parametric data and Mann-Whitney test for comparison of non-parametric data between the two groups. Multivariate analysis using regression model was applied to find factors independently associated with global improvement. A P value of <0.05 was considered significant in all analyses. BODY.RESULTS: During the study period 30 children were included in each group (mean age = 5.0 ± 2.9 years, 55% female); all of them completed the study [Figure 1]. The two groups were similar in age, gender, and baseline symptoms except stool frequency which was slightly lower in the control group (P = 0.024) [Table 1]. After the study, there was a significant improvement in most symptoms of constipation in both the synbiotic and control groups. However, improvements were greater in the synbiotic group regarding defecation frequency (even after controlling for baseline difference by ordinal regression test), straining, sense of incomplete evacuation, and the frequency of fecal incontinence (P < 0.05 to <0.001) [Table 2]. According to the SGI scale, symptoms were moderately and significantly improved in 13.3% and 86.6% of the subjects respectively, in the symbiotic group, while in the control group, improvement of symptoms was moderate in 36.6% and significant in 40% of subjects, which showed a significantly better global improvement in the synbiotic group (P < 0.001) [Table 2]. Figure 1Patients flow diagram Table 1Comparison of demographic data and baseline clinical characteristics between the two groups Table 2Comparison of symptoms at the end of intervention between the two groups BODY.MULTIVARIATE ANALYSIS: According to a difference between the two groups in baseline defecation frequency, we conducted a multivariate analysis to find factors associated with SGI results by controlling age, gender, kind of intervention, and baseline symptoms. Among the mentioned factors, only receiving synbiotic plus mineral oil compared with mineral oil alone was significantly associated with more global improvement (estimate coefficient = 2.359, CI95%: 0.862 to 3.857, P = 0.002) [Table 3]. Table 3Regression ordinal model on possible predictors of global improvement BODY.SIDE-EFFECTS: No severe side-effect was reported in any group. Reported side-effects were bloating (3.3%), diarrhea (3.3%), mild epigastric pain (3.3%), vomiting (3.3%), and abdominal pain plus itching and scar in the anal region in one case (3.3%). No side-effects were reported in the intervention group. There was no significant difference between the two groups in this regard. BODY.DISCUSSION: The aim of the present study was to evaluate the effectiveness of adding probiotic to the conventional treatment of childhood functional constipation. According to the results, adding a synbiotic which contained Lactobacillus Sporogenes to mineral oil resulted in more improvement in several symptoms of constipation compared with mineral oil alone. These results were also observed by the global improvement scale. Multivariate analysis with control of baseline symptoms and age and gender also approved these results. Limited data are available on the efficacy of probiotics in the treatment of constipation in children. In one study conducted by Bekkali and colleagues, children with functional constipation received a mixture of probiotics composed of Bifidobacteria and Lactobacili for 4 weeks. Results showed that the average of defecation frequency increased from 2/week at the beginning to 3.8/week at the end of the therapy. In contrast to our results, in the study by Bekkali et al., no significant change was observed in stool form. However, similar to our results, fecal incontinence decreased and no important side-effect was observed.[14] In another study by Bu and colleagues, children with constipation were allocated to three groups of Lactobacillus casei rhamnosus, Magnesium Oxide, and placebo. The results of this study showed that the probiotic was as effective as the magnesium oxide, while it did not have its possible side-effects.[15] In a study conducted by Khodadad and colleagues, children with constipation received Paraffin (1.5 ml/kg/d), synbiotic composed of Lactobacillus and Bifidobacteria, or a combination of synbiotic and Paraffin. The results showed that defecation frequency increased significantly in the combination group compared with the paraffin alone. But no beneficial effect was observed for stool form, incontinence, or painful defecation. Similar to our study, no side-effect was reported in the combination group while leakage of paraffin was a reported side-effect in those who received paraffin alone.[16] In contrast to the above mentioned trials showing the beneficial effects of probiotics in the treatment of childhood constipation, Banaszkiewicz and colleagues did not find beneficial effects of adding Lactobacillus GG to Lactose 70% over Lactose alone.[17] Differences among the results of available reports could be due to differences in the probiotic that was used; some studies used mixture compound while others used a single-strain probiotic. Therefore, further trials are needed in this regard. Available reports, however, are in favor of using probiotics as an adjunctive to other conventional therapies of childhood constipation. The possible mechanisms by which probiotics are beneficial in the treatment of constipation are not well studied. Available reports are focused on colonic transit time; however, more studies are needed in this regard.[8] There are some limitations of our study. There was a difference between the two groups in baseline characteristics possibly because of alternate allocation which is not qualified as randomization; however, we conducted a multivariate analysis to control the covariates and the results confirmed the beneficial effects of adding synbiotic to mineral oil. Also, it was ethically not possible to run a placebo-controlled group which received placebo alone; however, it was possible to add a placebo in the mineral oil group and our study has an important limitation in this regard. Therefore, our study was not blinded and the possibility of investigator bias exists. And finally, we had no follow-up to investigate the long-term effects of the intervention. BODY.CONCLUSIONS: The results of the present study showed that adding a synbiotic containing Lactobacillus Sporogenes to the routine treatment (mineral oil) of constipation in children can significantly increase improvement in symptoms, without specific side-effects. Further studies evaluating and comparing other synbiotic agents, either alone or along with other standard treatments, single or mixture of probiotics, and studies with longer follow-ups are warranted. Also, studies are needed on the possible mechanisms of action of probiotics in this regard.

TITLE: Continuous Right Thoracic Paravertebral Block Following Bolus Initiation Reduced Postoperative Pain After Right-Lobe HepatectomyA Randomized, Double-Blind, Placebo-Controlled Trial ABSTRACT.BACKGROUND AND OBJECTIVES: We hypothesized that continuous right thoracic paravertebral block, following bolus initiation, decreases opioid consumption after right-lobe hepatectomy in patients receiving patient-controlled intravenous analgesia with sufentanil. ABSTRACT.METHODS: Patients undergoing right-lobe hepatectomy with a right thoracic paravertebral catheter placed at T7 30 minutes before surgery were randomly assigned to receive through this catheter either a 10-mL bolus of 0.2% ropivacaine before emergence, followed by a continuous infusion of 6 mL/h for 24 hours (PVB group), or saline at the same scheme of administration (control group). All patients were started on patient-controlled intravenous analgesia with sufentanil in the postanesthesia care unit. The primary outcome measure was total sufentanil consumption during the first 24 postoperative hours. P = 0.05 was considered as significant. For the multiple comparisons of data at 5 different time points, the P value for the 0.05 level of significance was adjusted to 0.01. ABSTRACT.RESULTS: Sixty-six patients were assessed for eligibility, and a PVB catheter was successfully placed for 48 patients. Data were analyzed on 22 patients in group PVB and 22 patients in the control group. The cumulative sufentanil consumption in the PVB group (54.3 ± 12.1 μg) at 24 postoperative hours was more than 20% less than that of the control group (68.1 ± 9.9 μg) (P < 0.001). There was also a significant difference in pain scores (numerical rating scale) between groups, where the PVB group had lower scores than did the control group at rest and with coughing for the first 24 hours (P < 0.001). ABSTRACT.CONCLUSIONS: Continuous right thoracic paravertebral block, following bolus initiation, has an opioid-sparing effect on sufentanil patient-controlled intravenous analgesia for right-lobe hepatectomy patients and reduces numerical rating scale pain scores at rest and with coughing in the first 24 postoperative hours. Epidural analgesia has commonly been recommended following major upper abdominal surgery in the past,1 but this recommendation is controversial.2 Hemostatic deficiencies after hepatectomy may increase the bleeding risk associated with the use of thoracic epidural analgesia and delay epidural catheter removal.3 Epidural analgesia has been independently associated with increased risk of blood transfusion and administration of significantly greater volumes of intravenous fluid following liver resection.3 Balancing the advantages of optimal analgesia against the risks of current techniques requires additional clinical evidence in patients undergoing hepatectomy. Paravertebral nerve block (PVB) was first described by Sellheim of Leipzig in 1905 as an alternative to central neural blocks for obstetrics, particularly cesarean delivery. Paravertebral nerve blocks can provide high-quality analgesia for patients undergoing many types of surgery in the thoracic, abdominal, and pelvic regions and also for those suffering from trauma pain and chronic pain. The effect of continuous intraoperative and postoperative paravertebral block after right-lobe hepatectomy has been described in 2 patients, but the study did not include a control group.4 In this prospective, double-blind, randomized, placebo-controlled trial, we tested the hypothesis that continuous thoracic paravertebral block, following bolus initiation, reduces opioid consumption during the first 24 hours after right-lobe hepatectomy in patients receiving patient-controlled intravenous analgesia (PCIA) with sufentanil. The present study is reported according to the CONSORT (Consolidated Standards of Reporting Trials) statement. BODY.METHODS: BODY.PATIENT RECRUITMENT: This is a prospective, randomized, subject- and assessor-blinded, parallel-group, and placebo-controlled trial comparing a continuous right thoracic paravertebral block at T7 versus control group (saline) on postoperative opioid consumption after right-lobe hepatectomy for hepatocellular carcinoma. According to our local standards, the indications for all liver resections include tumor size of 5 to 10 cm; no intrahepatic or distant metastasis; no tumor thrombus in the portal vein, hepatic vein, vena cava, or bile duct; no invasion of the diaphragm or surrounding tissues; no rupture or bleeding of the tumor; indocyanine green retention rate at 15 minutes less than 15%; a remnant liver volume/standard liver volume ratio of greater than 50% in patients with liver cirrhosis and greater than 35% in patients without liver cirrhosis; Child-Pugh class A or B liver function, normal bilirubin concentration; and no previous liver resection. After receiving approval from the local institute’s ethics committee, registration with Clinical Trials (NCT01691937), and written informed consent from the patients, the study was conducted from September 2012 to February 2013 at Tongji Hospital in Wuhan, China. Patients were excluded if any of the following applied: a known allergy to the drugs being used, coagulopathy, on anticoagulants, analgesic intake, history of substance abuse, participation in the investigation of another experimental agent, inability to properly describe postoperative pain to investigators (eg, language barrier, neuropsychiatric disorder), or body mass index of 30 kg/m2 or greater or 15 kg/m2 or less. All patients were informed about the postoperative analgesia routine on the day before surgery and were instructed on how to express pain intensity with use of the numerical rating scale (NRS), where 0 indicates no pain, and 10 indicates the most severe pain. The same surgical team performed all surgical procedures through a subcostal incision. BODY.RANDOMIZATION: Simple randomization was done by independent research staff using opaque sealed envelopes, 24 for each group, indicating group assignment and describing the anesthetic protocol. A right thoracic paravertebral catheter was placed at the T7 level 30 minutes before surgery by an experienced senior consultant (W.M.). After confirmation of a successful paravertebral block, according to predefined criteria, patients were randomly assigned to the continuous PVB group or control group by research staff, and the PVB catheter was connected to an infusion pump containing either ropivacaine or normal saline accordingly. The infusion pumps are indistinguishable in appearance, and therefore the patients, the intraoperative caregiver (B.N.), the research staff assessing postoperative outcomes (H.C.), the acute pain service team, and the surgical team were blinded to group assignment. Unmasking did not occur until statistical analysis was complete. BODY.ANESTHETIC TECHNIQUE: A preoperative pain score at rest was recorded during the preoperative visit. No preoperative medications were administered. Upon arrival in the operating room, standard monitoring, including electrocardiography, noninvasive blood pressure, and pulse oximetry, was established. Surface electrodes for the Narcotrend Monitor (version 4.0; MonitorTechnik, Bad Bramstedt, Germany) were applied to the patient’s forehead, and the Narcotrend index was monitored. After placement of a peripheral venous catheter, a lactated Ringer’s infusion was started. Before anesthesia, all patients had continuous right thoracic paravertebral block at T7 performed under ultrasound guidance with a SonoSite M-Turbo transportable ultrasound device and a 38-mm linear 6- to 13-MHz ultrasound transducer (SonoSite M-Turbo; SonoSite Inc, Bothell, Washington) in the left lateral position via an in-plane needle insertion approach. Intravenous fentanyl and midazolam were titrated for patient comfort. After standard skin disinfection, the skin and periosteum of the rib were anesthetized with 1% lidocaine. The transducer was positioned in a transverse and partial oblique position to the vertebral column, parallel to the rib. At a point about 6 cm lateral to the midline, a prefilled 18-gauge, 10-cm Tuohy needle (PlexoLong Nanoline; Pajunk Inc, Geisingen, Germany) was inserted and oriented into the T7 paravertebral space between the internal intercostal membrane and the pleura under the real-time ultrasound guidance as described by Renes et al.5 After injection of 5 mL of 0.9% saline, an open-tip, single-orifice, styletted catheter (PlexoLong Nanoline; Pajunk Inc) was advanced 3 to 5 cm medially into the paravertebral space. After gentle and repeated aspiration, 1% lidocaine 15 mL was administered via the catheter over a period of at least 60 seconds. Fifteen minutes after block, sensory assessments were performed with a pinprick test, and the block level was recorded. The PVB was considered successful when subjects experienced a decreased sensation to pinprick in 3 or more adjacent dermatomes corresponding to the site of injection.6 Subjects with a successful catheter placement and nerve block onset per protocol were retained in the study. If the desired block was not achieved, we offered the patient the choice of reinsertion of the PVB catheter or to be excluded from the study. The properly located catheters were connected to a programmable portable, electronic infusion pump immediately before surgery. A nurse who was not otherwise involved in the study prepared and released the infusion pump containing blinded study medication with either 0.2% ropivacaine for the PVB group or 0.9% saline for the control group. In order to avoid inadvertent unmasking of allocation, the extent of sensory block was not tested during the postoperative period. In both groups, the patient was then turned supine, and general anesthesia was induced with fentanyl 3 to 4 μg/kg and propofol 1.5 to 2 mg/kg. Tracheal intubation was facilitated with 0.6 to 1 mg/kg rocuronium. The patients were mechanically ventilated with oxygen-air mixture maintaining the end-tidal CO2 at 4.5 to 5.0 kPa. Anesthesia was maintained by remifentanil and sevoflurane to keep the Narcotrend index reading between 40 and 60, and the systolic arterial pressure and heart rate within ±20% of baseline values during the procedure. Atracurium was administered for muscle relaxation as needed. The patient’s nasopharyngeal temperature was maintained between 36°C and 37.5°C by WarmTouch. All patients received a standard right-lobe hepatectomy for hepatocellular carcinoma by a J-shaped right subcostal incision. The J-shaped incision consisted of a right subcostal incision with a mediocranial extension to the xiphoid process. The right lateral extension was variable but comprised transection of the oblique abdominal musculature.7 Twenty minutes before the end of surgery, all patients received 1 to 2 μg/kg fentanyl titration and tropisetron 2 mg intravenously. At the same time, the PVB group had a 10-mL bolus of plain 0.2% ropivacaine via the paravertebral catheter by infusion pump, followed by an infusion of 0.2% ropivacaine at 6 ml/h. The same volume of 0.9% saline was injected and infused in the control group by infusion pump. At the end of surgery, the patient’s trachea was extubated when response to verbal commands, spontaneous respiratory rate exceeding 12 breaths/min, and end-tidal carbon dioxide partial pressure less than 45 mm Hg were observed. They were then transferred to the postanesthesia care unit (PACU). BODY.POSTOPERATIVE CARE AND PAIN ASSESSMENT: Upon PACU arrival, rest pain was evaluated by nurses using NRS (0–10) every 10 minutes. All patients received intravenous sufentanil (2–5 μg) titration at 10-minute intervals for NRS scores greater than 3 in PACU. Once an NRS score of 3 or less had been achieved, spontaneously or after a titration of sufentanil, patients were connected to a patient-controlled analgesia device (BCDB-200; BCM, Shanghai, China) set to deliver 1 μg sufentanil as an intravenous bolus with a 10-minute lockout interval and a continuous background infusion of 1 to 2 μg/h and a maximal permitted dosage of sufentanil 8 μg/h. An acute pain service team was responsible for maintenance of the PCIA pump according to local standard. Transition from PACU to surgical ward was considered safe when the patient had achieved a modified Aldrete score of greater than over equal to 9 for at least 10 minutes and physiological function to near preanesthetic levels. Pain at rest and on coughing was recorded for each patient using NRS (0–10) at 1, 4, 8, 16, and 24 hours after PACU discharge by the research staff (H.C.). Rescue opioid with intravenous tramadol was provided by acute pain service team when needed. Sedation was recorded using Ramsay Sedation Scale. Excessive sedation was defined as a Ramsay Sedation Scale value of 5 or 6 requiring the administration of naloxone. In the presence of nausea or vomiting, tropisetron 2 mg (maximum dose, 5 mg/d) intravenously was given and repeated if nausea persisted. The data of cumulative dose of sufentanil given postoperatively (titration and patient-controlled analgesia during the 24-hour observation period) and PCIA pressing frequency were recorded. Postoperative nausea and vomiting events during the first 24 hours were also recorded. After 24 hours, patients were asked to rate their overall satisfaction using an NRS, where 0 represents totally unsatisfied, and 10 represents completely satisfied. The infusion of ropivacaine in the PVB group and saline in the control group was stopped 24 hours after the surgery, and the catheter was removed. The PCIA continued until 48 hours after the surgery in both groups. BODY.STATISTICAL ANALYSIS: The primary outcome measure was total sufentanil consumption in the first 24 postoperative hours. Previously unpublished data including 132 patients from April 2012 to July 2012 provided by the acute pain service team in our institution showed a mean and SD of cumulative sufentanil consumption over the first 24 hours after right-lobe hepatectomy to be about 70 and 10 μg, respectively. In order to have 95% power to detect a 20% reduction of sufentanil consumption at an α level of 0.01, we needed 20 patients per study group. To account for any patient dropouts or missing data, we planned to enroll 24 patients per study group. Descriptive statistics were computed for all study variables. Normally distributed data were analyzed using 2-tailed Student t test. Repeated-measures analysis of variance (ANOVA) was used to compare measurements over time (cumulative sufentanil consumption and PCIA pressing frequency). If there was a statistical difference (P < 0.05) between the 2 groups by repeated-measures ANOVA, Student t test was used to compare the data at each time point. The non–normal distributed NRS data are displayed in box-and-whisker plots. The box extends from the upper quartile to the lower quartile and is marked by a quadrate at the median of the data. The whiskers are determined by the upper and lower data point values not including outliers and extremes. Outliers (>1.5 box lengths) and extremes (>3 box lengths) are represented by circles and asterisks, respectively. All NRS data including outliers and extremes were included in final analysis. Differences of NRS scores at each time point between the 2 groups were analyzed using the Mann-Whitney U test. Categorical data were analyzed using the χ2 or Fisher exact test, where appropriate. The data are reported as mean ± SD, mean (range), median (first third quartiles), or percent, as appropriate. P = 0.05 was considered as significant. The Bonferroni correction was performed on the raw P value where applicable. For the multiple comparisons of NRS, sufentanil consumption, and frequency of PCA data at 5 different time points, the P value for the 0.05 level of significance was adjusted to 0.01. All analyses were performed using SPSS (version 12; SPSS Inc, Chicago, Illinois). BODY.RESULTS: A total of 66 patients were assessed for eligibility, 9 patients did not meet inclusion criteria, and 6 patients refused to participate; PVB failed in 3 patients. A PVB catheter was placed successfully in 48 patients, 4 patients were excluded from final analysis because of catheter obstruction (n = 1), change of operative plan (n = 2), and unplanned postoperative mechanical ventilation (n = 1), respectively. Data were analyzed on 22 patients in each group, as shown in Figure 1. The characteristics of patients, duration of surgery, intraoperative variables, and liver function test indices 24 hours before and after surgery in the paravertebral and control groups are shown in Table 1. The baseline characteristics and intraoperative variables were similar in both groups. FIGURE 1CONSORT patient flow diagram. TABLE 1Patients’ Characteristics, Duration of Surgery, Intraoperative Variables, and Some Indexes of Liver Function 24 Hours Before and After Surgery in the PVB and Control Groups The time-matched cumulative sufentanil consumption and PCIA pressing frequency after surgery are shown in Figures 2 and 3. The cumulative sufentanil consumption and PCIA pressing frequency at each time point for the control group was significantly higher than the paravertebral group as indicated in Figures 2 and 3. Cumulative sufentanil consumption in the PVB group (54.3 ± 12.1 μg) at 24 postoperative hours was reduced by 20.2% compared with the control group (68.1 ± 9.9 μg), P < 0.001. FIGURE 2Cumulative sufentanil consumption in the first 24 postoperative hours. Data are presented as mean and SD. Differences of cumulative sufentanil consumption at each time point between the 2 groups were compared with repeated-measures ANOVA followed by Student t test. The Bonferroni correction was performed on the raw P value. Patients in the paravertebral group consumed less sufentanil than did patients in the control group at each time point. FIGURE 3Cumulative frequency of PCIA in the first 24 postoperative hours. Data were presented as mean and SD. Differences of cumulative frequency of PCIA at each time point between the 2 groups were compared with repeated-measures ANOVA followed by Student t test. The Bonferroni correction was performed on the raw P value. The cumulative frequency of PCIA at each time point in the control group was more compared with the paravertebral group. Pain scores within 24 postoperative hours at rest and with coughing are shown in Figures 4 and 5, respectively. The pain intensity at rest and with coughing reported with the NRS score was significantly lower in the paravertebral group as compared with the control group for each time point. The greatest difference in NRS score was found at 1 hour postoperatively in which the median (first third quartiles) NRS score on coughing was 1 (1–2) in the PVB group and 3 (3–4) in the control group (P < 0.001). FIGURE 4Pain scores at rest in the first 24 postoperative hours. The NRS data are displayed in box-and-whisker plots. Median (quadrate in the box), interquartile range (box), and range not including outliers (error bars) are shown. Outliers (>1.5 box lengths) and extremes (>3 box lengths) are represented by circles and asterisks, respectively. Differences of NRS scores at each time point between the 2 groups were analyzed using the Mann-Whitney U test. The Bonferroni correction was performed on the raw P value. The NRS scores at rest at each time point in the control group were higher compared with the paravertebral group. NRS (0–10), where 0 indicates no pain, and 10 indicates the most severe pain. FIGURE 5Pain scores on coughing in the first 24 postoperative hours. The NRS data are displayed in box-and-whisker plots. Median (quadrate in the box), interquartile range (box), and range not including outliers (error bars) are shown. Outliers (>1.5 box lengths) and extremes (>3 box lengths) are represented by circles and asterisks, respectively. Differences of NRS scores at each time point between the 2 groups were analyzed using the Mann-Whitney U test. The Bonferroni correction was performed on the raw P value. The NRS scores on coughing at each time point in the control group were higher compared with the paravertebral group. NRS (0–10), where 0 indicates no pain, and 10 indicates the most severe pain. The rescue analgesia, complications, satisfaction scores, and length of postoperative hospital stay after surgery of patients in both groups are shown in Table 2. The incidence of vomiting, bloating, and excessive sedation and the length of hospital stay were comparable between the groups. The patient satisfaction score was 8.1 ± 1.0 in the paravertebral group and 6.7 ± 0.6 in the control group (P < 0.001). One patient in the control group was documented to have an episode of respiratory rate below 8 breaths/min. The PCIA background dose was reduced to 1 μg/h sufentanil, and the respiratory rate returned to an acceptable level. TABLE 2Rescue Analgesia, Complications, Satisfaction, and Length of Hospital Stay After Surgery of Patients in the PVB and Control Groups Loss of pinprick sensation involved a mean of 4.6 (range, 3–7) dermatomes with upper and lower limits of T4 and T11 in the PVB group. The sensory loss to pinprick was 4.8 (range, 3–8) dermatomes, with upper and lower limits of T4 and T11 in the control group. There was no significant difference in extent of somatic block between the 2 groups following the 15-mL lidocaine testing injection (P = 0.618). BODY.DISCUSSION: In this prospective, randomized, double-blind, placebo-controlled study, a continuous unilateral thoracic PVB, following bolus initiation, produced an opioid-sparing effect within the first 24 hours after right-lobe hepatectomy. Patients receiving continuous thoracic PVB reported significantly lower pain scores than did those with sufentanil PCIA alone. Our results are consistent with previous findings showing that thoracic PVB reduced the severity of postoperative pain or opioid consumption following both minor surgeries such as thoracoscopic surgery,8 breast cancer surgery,9 and major surgeries such as thoracotomy.10 Paravertebral nerve block has been successfully used for pain management for cholecystectomy,11–13 radiofrequency ablation of a metastatic liver lesion,14 percutaneous transhepatic biliary drainage,15 and right-lobe hepatectomy.4 Richardson et al16 have demonstrated that thoracic PVB can abolish the intercostal somatosensory-evoked potentials. Liver capsule injury visceral pain was managed successfully with a continuous infusion of local anesthetic at the right T10 level through a paravertebral catheter,17 whereas common bile duct dilation–associated pain, mediated parasympathetically through the vagus, could not be alleviated by PVB.18 The liver and its capsule are innervated by sympathetic fibers arising from the thoracic sympathetic chain from the T7 to T11 level, and parasympathetic nerves derive from the vagus nerve.19 The right PVB-induced pain reduction following hepatectomy with right-sided subcostal incision may be due to blockade of somatic and sympathetic pain fibers originated from T5 to T11.19 The vagal nerve and contralateral sympathetic fibers are not targeted by unilateral PVB, so total visceral anesthesia may not be achievable. This may be considered a drawback of a unilateral PVB used for pain management of hepatobiliary surgeries. Bilateral PVB technique has been successfully used in thoracic, abdominal, and pelvic regions.20 But bilateral PVB using landmark techniques was found to double the likelihood of inadvertent vascular puncture and to cause an 8-fold increase in pleural puncture and pneumothorax.21 Recent modifications to PVB technique by ultrasound guidance may improve the safety of this technique. The extent of dermatomal spread of local anesthetics in the paravertebral space is variable and characterized by inconsistent somatic and sympathetic blocks. The mean extent of the somatic block was 4.6 and 4.8 dermatomes in the PVB group and control group, respectively, at 15 minutes after injection of 1% lidocaine 15 mL in the present study. Injections in a multilevel fashion may increase the extent of somatic and sympathetic block but would expose patients to additional risks related to punctures even under the ultrasound guidance. Further studies may be needed to test the clinical safety and efficacy of PVB at multiple levels for pain management after major abdominal surgeries. 22 Ultrasound-guided continuous thoracic PVB has been used for outpatient acute pain management of multilevel unilateral rib fractures.23 However, an unacceptably high misplacement rate of paravertebral catheters has been reported by using a landmark technique recently.24 Catheter displacement may be prevented by the introduction of coiled catheters into clinical practice.25 Because of ethical issues and safety consideration, we have not enrolled patients with impaired coagulation functions that contraindicated epidural analgesia. Common concerns with the PVB are the potential risk for pneumothorax and vascular or neural injury. Paravertebral nerve blocks are technically easy to learn with a high success rate of 89.9% to 93.9% and a relatively low rate of over all complications (5%) and serious complications such as pneumothorax (0.5%).21,26 A recent meta-analysis including 18 randomized controlled trials demonstrated that PVBs, compared with epidural analgesia, are associated with less urinary retention, postoperative nausea and vomiting, and hypotension.27 These results were consistent with previous meta-analysis performed by Davies et al28 in 2006. In addition, the rates of failed technique were also lower in the PVB group than those in the epidural analgesia group.27 Paravertebral nerve block may offer an attractive alternative to the epidural technique for patients with local sepsis, coagulation disorders, preexisting neurological disorders, and abnormal thoracic vertebral anatomy. A comparison between the standard epidural analgesia and ultrasound-guided PVB would be meaningful and warrants further studies. Patients in the PVB group had longer length of stay, and this may be due to differences in the American Society of Anesthesiologists–Physical Status (ASA-PS) class, age, and blood loss. Although these differences between groups were not statistically significant, together they may have accounted for longer hospitalization. This study has several limitations. Although a difference in the primary end point was shown, we were unable to demonstrate a difference in secondary outcome measures because of the small sample size. According to previous studies, we arbitrarily defined block success as loss of pinprick sensation in 3 or more ipsilateral vertebral dermatomes.6 After liver resection, the possibility of extending the analgesic benefits of paravertebral analgesia beyond 24 hours with or without a more concentrated solution of local anesthetic warrants further investigation. Further larger-scale studies are required to evaluate the effect of paravertebral block for right-lobe hepatectomy on clinically important outcomes including ropivacaine plasma levels, the incidence of postoperative complications, and chronic pain syndrome. Because sufentanil and ropivacaine are metabolized hepatically, elevated plasma levels can be seen after liver surgery.29,30 For safety concerns and in the absence of measurement of plasma levels of these medications, we limited the PVB infusion to 24 hours. This makes it more difficult to distinguish the effect of the initial ropivacaine PVB bolus from the continuous infusion on reducing pain scores and opioid consumption. It has been reported that single-shot PVB with 0.5% ropivacaine or 0.5% bupivacaine can produce 6- to 8-hour analgesic effects after thoracoscopic procedures.31,32 At the end of surgery, the 10-mL bolus of 0.2% ropivacaine prior to active infusion may have produced a significant opioid-sparing effect for an indeterminate number of hours out of the first 24 postoperative hours. The pain alleviating and opioid-sparing effects of PVBs in the first 24 postoperative hours demonstrated by our study may be the results of both the initiation bolus injection and the following continuous infusion of 0.2% ropivacaine.33,34 The present study aimed to test the hypothesis that a continuous PVB protocol (bolus initiation followed by continuous infusion of 0.2% ropivacaine) can reduce opioid consumption during the first 24 hours after right-lobe hepatectomy. We are unable to separate the effect of the initial bolus from the continuous infusion without having avoided the bolus, or running the infusion long enough to be certain there is no residual effect from the initial bolus. Obviously, keeping PVB catheter after single-dose PVB allows feasible rescue bolus injection or continuous infusion of local anesthetics when needed. Comparing the analgesic effects of bolus PVB, continuous PVB without bolus initiation, and bolus initiation followed by continuous infusion is not the primary interests of the present work. This issue clearly warrants further studies. Compared with patients from Western countries, the patients in our study have smaller body size, younger age, and lower ASA-PS scores. This unique demographic profile may impair the generalizability of our results, and further studies are needed to confirm the reproducibility of our technique in more overweight and/or more debilitated patients. In conclusion, this is the first prospective randomized, double-blind, placebo-controlled study evaluating the analgesic efficacy of continuous thoracic paravertebral analgesia in patients undergoing right-lobe hepatectomy. Although thoracic epidural analgesia and interpleural analgesia after hepatectomy have been reported, the use of paravertebral analgesia for hepatectomy patients has rarely been reported.4,35 Our data demonstrated that continuous thoracic paravertebral block, following bolus initiation, is an effective procedure to improve pain treatment after right-lobe hepatectomy and could be incorporated into multimodal analgesic regimens for right-lobe hepatectomy with a right-sided subcostal incision.

TITLE: Men perform comparably to women in a perspective taking task after administration of intranasal oxytocin but not after placebo Oxytocin (OT) is thought to play an important role in human interpersonal information processing and behavior. By inference, OT should facilitate empathic responding, i.e., the ability to feel for others and to take their perspective. In two independent double-blind, placebo-controlled between-subjects studies, we assessed the effect of intranasally administered OT on affective empathy and perspective taking, whilst also examining potential sex differences (e.g., women being more empathic than men). In study 1, we provided 96 participants (48 men) with an empathy scenario and recorded self-reports of empathic reactions to the scenario, while in study 2, a sample of 120 individuals (60 men) performed a computerized implicit perspective taking task. Whilst results from Study 1 showed no influence of OT on affective empathy, we found in Study 2 that OT exerted an effect on perspective taking ability in men. More specifically, men responded faster than women in the placebo group but they responded as slowly as women in the OT group. We conjecture that men in the OT group adopted a social perspective taking strategy, such as did women in both groups, but not men in the placebo group. On the basis of results across both studies, we suggest that self-report measures (such as used in Study 1) might be less sensitive to OT effects than more implicit measures of empathy such as that used in Study 2. If these assumptions are confirmed, one could infer that OT effects on empathic responses are more pronounced in men than women, and that any such effect is best studied using more implicit measures of empathy rather than explicit self-report measures. BODY.INTRODUCTION: Oxytocin (OT) is a highly conserved neuropeptide and an accumulation of its receptors are found in the amygdala (Loup et al., 1991). The amygdala is a structure that is part of the limbic system, associated with social behavior and emotion processing (Phelps and LeDoux, 2005), or more broadly with “relevance detection” (Sander et al., 2003). OT is involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis, and thus affects processes such as birth and breast-feeding in females and sexual mating, attachment and bonding in both sexes (see Carmichael et al., 1987, 1994; Carter, 1992; Altemus et al., 1995; Meston et al., 2004; Vignozzi et al., 2008). OT is also proposed to have anxiolytic effects (Uvnäs-Moberg, 1997; Heinrichs et al., 2003). Thus, OT acts centrally and peripherally as a central neurotransmitter/neuromodulator and a peripheral hormone in both males and females (Carter, 1998; MacDonald and MacDonald, 2010). In line with its anatomical and functional properties, OT is involved in human interpersonal information processing and behavior such as in enhancing prosocial judgments and behavior. For example, OT as compared to placebo administration (1) yielded higher trust in others (Kosfeld et al., 2005), (2) increased perceived attractiveness and trustworthiness of unfamiliar faces (Theodoridou et al., 2009), (3) increased charitable donations (Barraza et al., 2011), (4) promoted positive inferences about others' mental states (Domes et al., 2007), and (5) facilitated the identification of emotions regardless of valence (Lischke et al., 2012). Pointing to OT influences in clinical populations, individuals with autism benefited from OT administration by showing enhanced affective speech comprehension (Hollander et al., 2007), “mind-reading” (Guastella et al., 2010), and processing of social signals and social feedback (Andari et al., 2010). Likewise, in patients suffering from psychotic symptoms, OT administration improved performance in theory of mind tasks and perceptions of trustworthiness (Pedersen et al., 2011). Given OT's role in interpersonal information processing and behavior, it can be assumed that it also plays a role in empathy, that is, the ability to understand another's emotional perspective and to be personally affected by it in a way that mirrors the feelings of the individual (Eisenberg and Miller, 1987). Empathy is crucial to successful interpersonal skills and relations (Miller and Eisenberg, 1988; Batson, 1991; Eisenberg et al., 2002). According to most models, empathy consists of at least two components (Gladstein, 1983; Mahrer et al., 1994; Kerem et al., 2001). The first component accounts for the cognitive effort involved in considering another's viewpoint (i.e., perspective taking), and the second concerns vicarious emotional affective empathy (Davis, 1983; Hoffman, 2000; Blair, 2005), herein referred to as “affective empathy.” Both are required for normal “empathic ability” (Davis, 1983; Duan and Hill, 1996; Cialdini et al., 1997) and are shown to be related (Thakkar et al., 2009; Mohr et al., 2010; Thakkar and Park, 2010; Gronholm et al., 2012) and modulated by individual difference variables and personal experiences (Mohr et al., 2010; Cooper and Mohr, 2012). To date, examination of OT's role in empathy has been associated with two main pitfalls. Firstly, most studies have used self-report questionnaires, which lack accuracy and are prone to socially desirable responding Tierney and McCabe, 2001; Kämpfe et al., 2009; Gerdes et al., 2010; Taras et al., 2010. Secondly, research findings have been mixed, painting a rather unclear picture (Zak et al., 2007; Singer et al., 2008; Bartz et al., 2010; Hurlemann et al., 2010). On the one hand, studies show that OT administration enhances self-reported emotional (but not cognitive) empathy (Hurlemann et al., 2010) as measured by the Multifaceted Empathy Test (MET; Dziobek et al., 2008) as well as empathic accuracy (Bartz et al., 2010), that is, the ability to accurately rate others' feelings when they narrate emotional events, in particular in listeners that are not socially proficient. Also, OT as compared to placebo administration enhanced perspective taking ability and generosity toward others in an economic game; i.e., more money was transferred to partners after having imagined their perspective and considered their reaction to an offer (Zak et al., 2007). On the other hand, OT versus placebo administration exerted a null effect on emotional empathic responses to a romantic partner's pain, i.e., on self-reported unpleasantness ratings when considering the partner's experience of painful hand stimulation (Singer et al., 2008). These inconsistent findings may either be due to OT and empathy being unrelated, or problems with the self-report measurement of empathy. We here consider these possibilities. In two independent studies, we used comparable double-blind placebo-controlled between-subject designs to assess healthy individuals' empathy as a function of nasal OT administration. In study 1, we provided participants with a vignette in which a person's unfortunate plight was described. Participants rated their empathic feelings toward the individual (see e.g., Coke et al., 1978; Batson et al., 1989; Mikulincer et al., 2001); thereby directly linking the self-reported empathic response to an individual's plight. In study 2, we used a more implicit strategy by assessing reaction times for perspective taking in a computerized task. In this task, participants see back-facing and front-facing human figures sequentially on the computer screen and have to match the own perspective with the one of the figure (e.g., Mohr et al., 2010; Thakkar and Park, 2010; Gardner et al., 2012). Matching the own body position with that of a front-facing figure is cognitively more challenging than matching it with a back-facing figure as reflected in enhanced reaction times (see Figure 1A). This task has been used to examine various questions on cognitive functioning such as those underpinning different forms of mental rotation (Ratcliff, 1979; Zacks et al., 1999), cognitive correlates of out-of-body experiences (Blanke et al., 2005; Easton et al., 2009; Braithwaite et al., 2011) and schizotypy (Mohr et al., 2006; Easton et al., 2009), to evaluate learning (Bailey et al., 2007), and spatial compatibility effects (Gardner and Potts, 2011). Empirical evidence showed that at least part of task performance variance is modulated by empathy. For instance, in this 3rd person perspective taking task (3PP-task) increasing self-reported empathy scores are negatively correlated with response speed (Thakkar et al., 2009) in women but also positively correlated with greater accuracy (Thakkar and Park, 2010) and reaction times (Mohr et al., 2010), in particular for individuals reportedly using a social rather than a spatial perspective taking strategy (Gronholm et al., 2012). Thus, if OT enhances empathy, irrespective of the assessment format chosen, we would expect that increased OT availability enhances individuals' empathy in both studies, leading to higher empathic concern ratings in study 1 and potentially faster reaction times in study 2. Yet, if explicit, self-report measures of empathy bias desirable responding, the effect of OT might not be observed in study 1, with the 3PP task in study 2 producing more pertinent results, at least statistically. Figure 1(A) Examples of figures used in the perspective taking task. From left to right: The first two frames illustrate front- and back- facing female figures, while the remaining two frames depict front- and back- facing male figures. (B) Demonstration of the perspective taking task procedure: The cross is presented first, followed by the coloured dot, followed by the figure. In addition to these hypotheses, we considered the role of participant sex. Some studies of OT effects support an enhancing role of OT for interpersonal behavioral responses in both sexes (for evidence of absence of sex-dimorphic effects of OT see Ditzen et al., 2009; Theodoridou et al., 2009, 2011; Alvares et al., 2010; Rockliff et al., 2011), but others yield direct evidence for potential sex-specific effects of this neuropeptide Fischer-Shofty et al., 2013; Theodoridou et al., 2013; for a recent review on the role of sex in individual responses to OT see MacDonald, 2012). It is worth noting that studies that have reported effects of OT specifically on empathy and/or perspective taking either recruited only men (Zak et al., 2007; Bartz et al., 2010) or found that OT selectively affected men (Hurlemann et al., 2010). In light of the above inconsistent evidence (Ditzen et al., 2009; Hurlemann et al., 2010), we considered the possibility that OT might exert sex-specific effects on empathy and perspective taking and tested an equal number of women and men. BODY.MATERIALS AND METHODS: BODY.PROCEDURE COMMON TO BOTH STUDIES: In both studies, we conducted two sessions: a baseline session performed by participants at home and a laboratory session for which participants came to the University. Before participation in either session we obtained written informed consent from each participant. We only tested participants who met our inclusion criteria: being a fluent English speaker, not having consumed any medication, or having any other medical reason why they should not receive OT. In the case of female participants, they could not be pregnant, or if post-birth, should not be breastfeeding. The research protocol in both studies was approved by the Faculty of Science Human Research Ethics Committee at the University of Bristol. We recruited participants through poster advertisement in and around university buildings and sent emails to various departments and posted on the university's jobs website. In the baseline session, participants provided demographic information (e.g., age, sex) and filled in self-report questionnaires such as the Major Depression Inventory (MDI; Bech, 1997); results from these measures can be found in Theodoridou et al. (2009) and Theodoridou et al. (2013). Participants sent the completed questionnaires to the experimenter. In the laboratory session at the local university, approximately one week later, participants were tested individually. They were instructed to abstain from alcohol, caffeine, and nicotine for 24 h before testing and from food and drink (except water) for 2 h before testing. When they arrived in the laboratory, each participant signed an informed consent form. The session lasted up to 2 h, with the actual task battery being assessed in the first 60 min after the waiting period, and the remaining time being used to guard for potential side effects. At the very beginning, participants were told that they would first receive a small dose of OT or a near identical looking and smelling placebo before being tested in various tasks. Information was also given about possible side effects associated with OT administration and participants were informed that they had the right to withdraw from the study at any time. In a double-blind procedure, participants were randomly assigned to self-administer a small intranasal dose of either 24 IU OT (Syntocinon Spray, Novartis, 3 puffs per nostril, each puff containing 4 IU OT), or placebo (containing the same ingredients, but OT, to the OT nasal spray). After a waiting period of 25–30 min, participants completed the task battery including the empathy vignette task (Study 1) and the 3PP-task (Study 2). Tasks were presented in two blocks, randomized in order. In study 1, the empathy scenario task was administered either 35 or 55 min after drug administration. In study 2, the 3PP-task was completed either 35 or 60 min after drug administration (see Theodoridou et al., 2009, 2011, 2013 for additional results from these studies). This time window is likely sensitive to OT effects; Gossen et al. (2012) showed that OT reaches its peak plasma level at approximately 30 min after a dose of 26 IU intranasal OT. Also note that the present protocol has led to established methods and findings in our laboratory before (see Theodoridou et al., 2009, 2011, 2013). In the laboratory sessions, we also assessed current mood, wakefulness, and calmness with the short form of the Multidimensional Mood State Questionnaire (Steyer et al., 1997). This 6-point scale consists of 15 items with answers ranging from “definitely not” to “extremely.” In Study 1, this measure was completed once, whereas in Study 2, it was completed twice, immediately before drug administration and immediately before testing. After testing, participants were instructed to guess the substance they had received1. Finally, participants were debriefed and offered either a monetary reward of £15 or experimental credit (or chocolate in a few instances). BODY.STUDY 1: BODY.PARTICIPANTS: Of the 96 participants (mainly students, mean age: 21.4 years, age range: 18–40 years), 51 (25 males) received OT, and 45 received placebo (23 males). As reported in our previous study (Theodoridou et al., 2009), any drug effect, sex effect, or interaction cannot be explained by participants' current affect, wakefulness or calmness. Likewise, independent samples t-tests showed no significant differences between drug groups in depression and trait anxiety (see Theodoridou et al., 2011 for details). BODY.EMPATHY TASK: Emotional reactions to another person's plight were assessed using a procedure similar to that employed previously (e.g., Coke et al., 1978; Batson et al., 1989; Mikulincer et al., 2001). More precisely, the experimenter read out a brief story about the plight of a university student who had recently lost her parents in a car accident. The full story read as follows: “Anna is 21 years old studying, on a full-time basis at the University of Bristol. A month ago her parents and older sister got killed in a car accident. At the moment she is desperately trying to take care of her surviving younger brother and sister while trying to finish her last year of BSc studies. If she does not complete her degree, she will not be able to earn enough money to support her brother and sister and will have to put them up for adoption. What is more, Anna has no relatives that can help her out.” Immediately afterwards, the experimenter read out 10 adjectives (taken from Batson et al., 1989; see below), each of which participants verbally rated on a 7-point visual analog scale according to how they felt while listening to the story (1—not at all felt, 7—very strongly felt). Participants were asked to bear in mind that the student was a mere acquaintance to them. We stressed this point because we wished to examine the effect of OT on prosocial behavior toward non-intimate others to avoid ceiling effects due to emotional closeness to the main character of the story. A digital voice recorder (Olympus; VN-2100PC) was used to record responses in this task. Half of the adjectives tap on empathic concern (i.e., other-oriented emotional empathy at the plight of others): sympathetic, soft-hearted, compassionate, tender, moved, and the other half tap on feelings of personal distress (i.e., self-oriented emotional reactions at the plight of others): alarmed, grieved, distressed, upset, and disturbed. We calculated mean scores for empathic concern responses and personal distress responses, separately (range of scores 1–7 with higher scores reflecting greater empathic concern and personal distress, respectively) to account for the possibility that these two dimensions are differently influenced by OT and/or sex. For instance, OT might increase empathic concern for a person in need and/or attenuate feelings of personal distress (Batson et al., 1987). OT might act anxiolytically (Carter et al., 2001; Heinrichs et al., 2001, 2003; Cardoso et al., 2012; de Oliveira et al., 2012; for reviews see Meyer-Lindenberg et al., 2011; Striepens et al., 2011; MacDonald and Feifel, 2012) decreasing discomfort and concern for one's own self. BODY.STUDY 2: BODY.PARTICIPANTS: Of the 120 participants (mainly students, mean age: 22.4 years, age range: 18–44 years), 60 (30 males) received OT, and 60 received placebo (30 males). As in study 1 (Theodoridou et al., 2009), we observed no influence of drug and/or sex on participants' current affect, wakefulness, or calmness. In more detail, following Domes et al.'s (2007) procedure, we calculated difference scores on the different mood measures affect, wakefulness and calmness for time 1 (pre drug administration) and time 2 (30 min post drug administration) by subtracting Mood at time 2 from Mood at Time 1. Thus, positive values reflect better mood at time 1 and negative values reflect better mood at time 2. We performed a multivariate ANOVA (MANOVA) on these difference scores with drug (OT, placebo) and participant sex (male, female) as between-subjects variables. There was no effect of drug on change in affect, F(1, 116) = 0.02, p = 0.90, wakefulness, F(1, 116) = 0.04, p = 0.85, and calmness, F(1, 116) = 0.06, p = 0.80. Similarly, no effect of participant sex was found on change in affect, F(1, 116) = 0.10, p = 0.75, change in wakefulness, F(1, 116) = 0.02, p = 0.90, and change in calmness, F(1, 116) = 1.71, p = 0.19, and no significant drug × participant sex interactions (all ps > 0.1). BODY.PERSPECTIVE TAKING TASK: Picture preparation. To make figures more realistic, we here refrained from using schematic drawings used before (Mohr et al., 2010, in press; Cooper and Mohr, 2012) and took photographs of an adult man and an adult woman of approximately the same height instead, both dressed in jeans and black T-shirts (see also Thakkar et al., 2009; Thakkar and Park, 2010). The photographs showed these adult models standing upright with the arms slightly outstretched to the right and left (see Figure 1A). Each of them was photographed in this same position from the front and the back. For each picture taken, the models were always wearing a black glove on the one hand and a brown glove on the other hand. The side of the black glove was once on the right and once on the left for both front-facing and back-facing positions. This counterbalancing resulted in eight possible photographs that were all of the same size (237 × 239 pixels), cropped and set against a white background (see Figure 1A for representative examples). For single trials, a centrally placed fixation cross appeared first for 800 ms followed by a centrally presented dot (diameter = 1.5 cm) for 1000 ms that was black in half of the trials and light brown in the remaining trials. After the disappearance of the dot, one of the pictures was presented centrally (visual angle ≈ 5° width × 6.1° height) until a response was provided, that is, the task was self-paced. Task procedure. First, participants received the written instruction that the following task would assess their empathic ability. The exact instruction was as follows: “This is a test of your ability to see the world from another person's perspective. Performance on this test reflects empathetic ability. Empathy is a social skill that is defined as being able to identify with, and understand what another person is perceiving, and to respond appropriately.” This instruction was based on previously used instructions (Massa et al., 2005) and was included to ensure that the task was introduced as a test of empathic abilities. Participants then received both a written and a verbal instruction to imagine being in the other person's shoes. Specifically, the instruction was: “Imagine you are in the person's body position. If the coloured circle is black, indicate which hand the black glove is on. If the coloured circle is brown, indicate which hand the brown glove is on. If the glove would be on your own left hand, press key ‘1.’ If the glove would be on your own right hand, press key ‘5.’ Please respond as quickly and accurately as possible, but always aim to take the other person's perspective first.” Following task instructions, a slide with the demonstration of the task procedure was presented for 6000 ms (see Figure 1B). Each picture was presented 10 times resulting in 80 experimental trials. The task was preceded by eight practice trials. We assessed the number of correct responses and the reaction times for correct responses. BODY.DATA ANALYSIS: In Study 1, data from one participant was excluded because this person responded especially slowly (average response time: 18 s). We also excluded scores (ratings) that were two standard deviations above or below the mean (1.51% of the data). A MANOVA test was carried out on mean empathic concern ratings and mean personal distress ratings, with drug (OT, Placebo) and participant sex (male, female) as between-subjects variables. One-sample tt-tests were carried out, on the empathic concern ratings and on the personal distress ratings separately for the sexes in each drug group. In Study 2, reaction times shorter than 200 ms and longer than 5000 ms were considered to be outliers and were dropped (Harris et al., 2002; Mohr et al., 2010). Incorrect trials were also discarded. The data of five participants were discarded because their error rates (ranging from 27.5% to 48.75%) were more than two standard deviations above the mean (Mean error rate = 7.84%, SD = 8.24). A mixed model ANOVA was carried out on the mean RT data, with drug (OT, placebo) and participant sex (male, female) as between-subjects variables, and target sex (male, female), position (front, back) as repeated measures. No statistical analyses were performed on accuracy data as the average error rate (%) after removal of the five outlying cases was very low (Total Mean = 6.59%; OT Mean: 6.68%; Placebo Mean: 6.49%). Pairwise post-hoc comparisons were based on Newman-Keuls tests. In both studies, we performed univariate ANOVA tests to examine potential age differences between the two drug groups and the two sex groups. The alpha level for all the statistical test results reported henceforth was set to 5% (0.05). BODY.RESULTS: BODY.STUDY 1: OT AND RATINGS OF ADJECTIVES SUBSEQUENT TO AN EMPATHY SCENARIO: BODY.PARTICIPANTS: The ANOVA on age showed no significant main effects [drug: F(1, 92) = 0.33, p = 0.57, participant sex: F(1, 92) = 0.003, p = 0.96] and no significant interaction, F(1, 92) = 0.01, p = 0.91. BODY.ADJECTIVE RATINGS: The MANOVA showed that OT (vs. placebo) had no effect on empathic concern, F(1, 90) = 0.16, p = 0.69, or personal distress ratings, F(1, 90) = 0, p = 0.99. A significant main effect of participant sex was found for empathic concern ratings, F(1, 90) = 4.18, p = 0.04, with females self-reporting greater empathic concern (M = 5.30, SD = 0.99) than males (M = 4.93, SD = 0.77). No effect of sex was found on personal distress scores, F(1, 90) = 1.91, p = 0.17. We did not observe any significant interactions [largest interaction effect: F(1, 90) = 1.41, p = 0.24]2. When comparing the two adjective rating scores against chance level (3.5), the one-sample t-tests showed that the mean empathic concern rating (M = 5.12; SD = 0.90) was significantly higher than chance level, t(93) = 17.38, p < 0.001, as was the mean personal distress rating (M = 3.83; SD = 1.27), t(93) = 2.52, p = 0.01. The same conclusion could be drawn when performing the same comparisons for the two sexes in each drug group, separately (see Table 1 for detailed results). The opposite conclusion could be drawn when performing the same comparisons for mean personal distress ratings, i.e., the mean adjective ratings scores were not different from chance level, apart from higher scores in female participants in the placebo group (see Table 1). Table 1Mean empathic concern ratings and their difference from chance level (3.5) for the two sexes in each drug group. Empathic concernt (p-value)Personal distresst (p-value)OT M 4.93 (0.64) 10.93 (<0.001) 3.80 (1.01) 1.47 (0.15) F 5.23 (0.89) 9.82 (<0.001) 3.86 (1.46) 1.24 (0.23) P M 4.93 (0.91) 7.39 (<0.001) 3.49 (1.18) −0.04 (0.97) F 5.39 (1.10) 8.03 (<0.001) 4.16 (1.35) 2.30 (0.03) Notes: standard deviations are in parentheses; OT, oxytocin; P, placebo; M, Male; F, Female; OT Male df = 23; P Male df = 21; OT Female df = 25; P Female df = 21. BODY.STUDY 2: OT AND PERSPECTIVE TAKING: BODY.PARTICIPANTS: The ANOVA on age showed no significant main effects [drug: F(1, 116) = 0.87, p = 0.35, participant sex: F(1, 116) = 1.11, p = 0.29] and no significant interaction, F(1, 116) = 0.02, p = 0.89. BODY.REACTION TIME ANALYSIS FOR THE 3PP-TASK: The ANOVA on mean reaction times for correct decisions showed a significant main effect of figure position, F(1, 111) = 124.03, p < 0.001, with front-facing figures eliciting longer reaction times (M = 1122.53, SD = 346.86) than back-facing figures (M = 913.93, SD = 255.64). In addition, the main effect of figure's sex, F(1, 111) = 31.68, p < 0.001, indicated that participants responded faster to female figures (M = 994.69, SD = 271.36) than to male figures (M = 1040.09, SD = 304.05). The main effects of drug, F(1, 111) = 1.8, p = 0.18, and participant sex, F(1, 111) = 1.35, p = 0.25, were both not significant3. We found significant 2-way interactions between figure position and participant sex, F(1, 111) = 9.03, p = 0.003, and figure position and target sex, F(1, 111) = 10.3, p = 0.004, and a significant 3-way interaction between drug, target sex, and participant sex, F(1, 111) = 5.7, p = 0.02. Post-hoc comparisons on these significant interactions showed for the figure position by participant sex interaction that male participants had faster reaction times than female participants for the front-facing condition (p = 0.04) (see Figure 2A). The same comparison for the back-facing condition was not significant (p = 0.92). Moreover, reaction times were significantly faster in the back- than front-facing condition for both male (p < 0.001) and female participants (p < 0.001). Post-hoc comparisons on the figure position by target sex interaction showed that female figures were responded to faster than male figures in the back-facing condition (p < 0.001) and front-facing condition (p = 0.04) (see Figure 2B). In addition, reaction times for back-facing figures were significantly faster than for front-facing ones for both male and female targets (all ps < 0.001). Figure 2Mean reaction times (A) of male and female participants to target figures in the back- and front-facing position, (B) to male and female target figures in the back- and front-facing position, (C) of male and female participants to target figures in the OT group and (D) the placebo group. Vertical bars indicate standard errors. Finally, to further elucidate the significant drug, target sex, and participant sex interaction, we performed 2-way ANOVAs, for the OT and the placebo group separately, with figure sex as a repeated measure and sex as a between subject-factor. The ANOVA for the OT group only showed the significant main effect on figure sex, F(1, 56) = 11.88, p = 0.001, i.e., that reaction times were faster for female than male figures. The main effect of sex, F(1, 56) = 0.14, p = 0.71, and the interaction, F(1, 56) = 1.96, p = 0.17, were both not significant (Figure 2C). The ANOVA for the placebo group, however, showed in addition to the main effect of figure sex, F(1, 54) = 24.58, p < 0.001 (male figures > female figures), a main effect of participant sex, F(1, 54) = 6.34, p = 0.01. Male participants responded faster than female participants (Figure 2D). Finally, the interaction was significant, F(1, 54) = 4.25, p = 0.04. Post-hoc comparisons showed that reaction times were faster for female than male figures in female (p < 0.001) and male (p = 0.045) participants. Moreover, male as compared to female participants showed a significant reaction time advantage for male figures (p = 0.02) that was only a statistical trend for female figures (p = 0.08). BODY.DISCUSSION: In two independent double-blind, placebo-controlled between-subject design studies we investigated whether the consumption of a single dose of OT affected the ability to empathize with another individual's unfortunate plight (Study 1) and the ability to mentally take the perspective of another person (Study 2). In study 1, we provided participants with a scenario designed to elicit empathy [modified version used by Coke et al. (1978); Batson et al. (1989)] and assessed two levels of self-reported empathy (empathic concern and personal distress) by having participants rate empathy relevant adjectives. In study 2, we recorded reaction times in a computerized 3PP-task to assess empathy more implicitly (e.g., Thakkar et al., 2009; Mohr et al., 2010, in press) and to also avoid the response biases often associated with self-report measures (Kämpfe et al., 2009). Given that OT has been previously shown to enhance interpersonal processes and behavior (for reviews see MacDonald and MacDonald, 2010; Bartz et al., 2011; Guastella and MacLeod, 2012); including empathy (Hurlemann et al., 2010) and related phenomena such as “mind-reading” (Domes et al., 2007; Guastella et al., 2010), we hypothesized that OT as compared to placebo administration would result in enhanced empathic responses (study 1) and speeded perspective taking (study 2). We also considered that this enhancement might be more prominent in study 2, because response biases associated with self-report measures might override the OT effect in study 1. Our main findings were that (1) the adjective ratings in study 1 (empathy scenario) did not reveal higher empathic responses in the OT as compared to the placebo group, and (2) sex differences in the 3PP-task (male over female participant advantage, in particular for male figures) observed in the placebo group were absent in the OT group. Before discussing these results and additional task findings, we would like to mention that neither study 1 nor study 2 found effects of OT administration on affect, wakefulness and calmness, indicating that the above reported behavioral effects of OT could not be attributed to or mediated by changes in mood. These findings are in agreement with previous studies that have reported null effects of OT on mood (Kosfeld et al., 2005; Domes et al., 2007; Fischer-Shofty et al., 2010; Lischke et al., 2011). BODY.OT AND EMPATHY RATINGS WHEN EXPOSED TO THE PLIGHT OF AN UNKNOWN PERSON: In study 1, the OT and placebo group provided comparable adjective ratings after hearing about an unknown person's plight. By inference the two groups reported comparable empathic reactions and personal distress when rating these adjectives. Contrary to previous findings supporting the role of OT in prosocial and affiliative behavior (Kosfeld et al., 2005; Theodoridou et al., 2009; Barraza et al., 2011), our results suggest that OT may not play a role in the experience of affective empathy. However, given that to be empathetic is a socially desirable trait and that participants spoke aloud their answers to the experimenter, we can assume that our methodology was prone to self-report biases (e.g., see Tierney and McCabe, 2001; Kämpfe et al., 2009; Gerdes et al., 2010; Taras et al., 2010). Our findings indicate that participants seemed inclined to provide high scores in these ratings: the average empathic concern rating was 5.12, thus, biased toward the highest possible score of seven. Our results show that this mean rating was significantly different to chance level 3.5 (i.e., mid-scale point), and that our female participants' self-reports of empathy deviated more strongly from this chance level than did male participants' self-reports. What is more, the rating of 5.12 appears high compared to values obtained in previous studies using similar (but not identical) designs, and in which seven was also the highest possible value [e.g., see Batson et al., 1988, studies 1 and 3 (3.81 and 3.88, respectively); Batson et al., 1989, study 2 (4.44 and 5.79)]. Consistent with sex differences observed by Batson et al. (1988, 1989), women scored higher in empathic concern than men (see also Baron-Cohen and Wheelwright, 2004; Hurlemann et al., 2010). As for personal distress ratings, the mean value here was lower than the value in a comparable study (e.g., in Batson et al., 1988, study 2). Given the overall caution with regard to self-report measures in this domain (Baron-Cohen and Wheelwright, 2004; Kämpfe et al., 2009), we suggest that empathic concern adjectives might tap onto sex stereotypes regarding emotions, i.e., women being more caring and higher in sympathy than men (Plant et al., 2000). By inference, such stereotypes may trigger stronger response biases and desirable responding than personal distress adjectives. Importantly, our results show no effect of drug administration on personal distress or empathic concern. Therefore, our findings fail to provide evidence for an anxiolytic and prosocial effect of OT, respectively, in the context of affective responses to an individual's plight. It is worth noting that in line with previous relevant studies (Batson et al., 1988, 1989; Mikulincer et al., 2001) we only tested affective responses to a woman's plight. Future studies should balance out for possible sex-specific effects and include a male target, although a faster reaction toward women is generally likely (see Lewin and Herlitz, 2002; Cellerino et al., 2004; Mohr et al., 2010) in line with our finding of speeded responses to female figures in study 2. To further understand the influence of sex on the link between OT effects on empathic abilities we tested an equal number of women and men. Studies in which individuals were provided with audio tapes narrating the plight of a needy person showed that women reported higher levels of empathy than men for the needy person (Batson et al., 1988, studies 1 and 3), and that reports of empathy for the needy person were relatively high (Batson et al., 1988, studies 1 and 3; Batson et al., 1989, study 2). Such sex differences in self-reports of empathy have often been documented, but could reflect a female tendency toward more socially appropriate responding relative to men (Eisenberg and Miller, 1987). In our study we did indeed observe that females' self-reports of empathy deviated more strongly from chance level than did males self-reports. However, no differential effect of OT was found in men's and women's self-reported empathy. BODY.OT AND REACTION TIMES IN A COMPUTERIZED 3PP-TASK: In study 2, we found that speed of response in the drug groups interacted with figure sex and participant sex. We observed that sex differences in the placebo group were absent in the OT group. More precisely, in both drug groups, we found that people responded faster to female than male figures. In the placebo group, we additionally observed that men responded faster than women, and that this sex difference was statistically significant for male figures and a statistical trend for female figures. Given that these sex differences are absent in the OT group, we infer that OT might have sex-dimorphic effects on this measure of perspective taking ability, an inference that is in line with a recent review by MacDonald (2012). The observation that male participants in the OT group responded as slowly as female participants in the OT group could further indicate that men adopted a comparable perspective taking strategy to the one used by women. This conjecture assumes that the women in our study, irrespective of whether they have received OT or placebo, adopt a relatively more time-consuming social perspective taking strategy when completing the 3PP-task as compared to the men in the placebo group. Evidence for this suggestion comes from research indicating that women experience greater rotational costs for front-facing figures (reflected in increased reaction times and decreased accuracy) than men when performing a 3PP-task (Mohr et al., 2010). In addition, further studies on computerized perspective taking ability indicate that social strategies might be more prevalent in women and object-based spatial strategies more prevalent in men, and that women find the 3PP-task more effortful, as reflected in lower accuracy rates (Kaiser et al., 2008). The link between faster reaction times and self-reported empathy seems to be most evident for women (Mohr et al., 2010), while no role of sex is reported in two further studies (Thakkar and Park, 2010; Gardner et al., 2012). Finally, studies using a similar (but not identical) perspective taking task to ours showed that women with high affective empathy scores are slower on perspective taking (Thakkar et al., 2009; but also see Thakkar and Park, 2010). If OT fosters social perspective taking in men, we could expect in future studies that a higher than normal OT availability may facilitate men's tendency to step into another person's shoes, a fundamental component of empathy (e.g., see Kaiser et al., 2008; Thakkar et al., 2009; Gardner et al., 2012). The present conclusion is relevant to empathy and OT researchers as it suggests that OT's beneficial effects might not be general but nuanced (Bartz et al., 2011), i.e., affecting men more strongly than women, at least in the context of social perspective taking. Alternatively, such beneficial OT effects might be evident in those who are less socially proficient, which men are thought to be (see also Bartz et al., 2010). We suggest that the above findings on OT effects in men are not an artifact of overall or aberrant performance in our study population, because we replicated previous behavioral findings using slightly modified versions of the current 3PP-task. Firstly, reaction times were faster for back-facing than front-facing pictures (see also e.g., Arzy et al., 2007; Mohr et al., 2010, in press; Thakkar and Park, 2010; Cooper and Mohr, 2012) indicating that participants performed mental transformations. This finding is in line with previous reports regarding the mental rotation of objects (Shepard and Metzler, 1971; Wohlschläger and Wohlschläger, 1998), body parts (Cooper and Shepard, 1975; Parsons, 1987; Bonda et al., 1995; Petit et al., 2003; Seurinck et al., 2004), and perspective taking tasks (e.g., Kaiser et al., 2008; Rilea, 2008) which report longer reaction times when the position of a stimulus (or own current body position) does not match the position of the target stimulus. Moreover, mental perspective transformations for female figures were faster than those for male figures, an advantage that was specific to back-facing figures, supporting previous observations (see Mohr et al., 2010). BODY.CONCLUSIONS AND POSSIBILITIES: Two studies examined the differential effects of OT on empathic responses, once using self-report ratings after having heard the story of an unknown person's unfortunate plight (study 1) and once using reaction times in a computerized 3PP-task (study 2). The major findings were that while OT as compared to placebo administration did not enhance self-reported empathic concern toward others (study 1), it showed that a male over female advantage in the 3PP-task that was evident in the placebo group was absent in the OT group (study 2). This finding is suggestive of a potential strategy change (purportedly more social than spatial performance strategy, see rationale in more detail above) in men after consuming OT as compared to placebo. Such a facilitation of social perspective taking might already be present in women, regardless of which of the two drugs were consumed. Thus, additional OT availability might affect men but not women in the 3PP-task. This conjecture, if supported in the future, might be relevant to sub-populations low in appropriate social abilities, such as individuals with alexithymia, social anxiety disorder, and schizophrenia (Caldwell et al., 2009; Guastella et al., 2009b; Bartz et al., 2010; Feifel et al., 2010; Rubin et al., 2010; Luminet et al., 2011; Pedersen et al., 2011; Hall et al., 2012) as it has already been shown in autism spectrum disorders (Andari et al., 2010; Guastella et al., 2010). Given the conjectural nature of our conclusions, future studies should verify the strategy participants employ in the 3PP-task, e.g., examine whether women are slower because they use a social perspective strategy while men (at least without pharmacologically enhanced OT availabilities) use a spatial perspective taking strategy (Gardner et al., 2012; but also see Gronholm et al., 2012). Indeed, one could also reason that OT administration hindered a spatial strategy that might have been favored by men, without having to necessarily facilitate a social strategy. While this possibility cannot be excluded, we consider our initial explanation to be more likely because men and women seemingly used different strategies in perspective taking including mental rotation (e.g., Weiss et al., 2003; Kaiser et al., 2008), and women with higher self-reported empathy were found to slow down in perspective taking, presumably because they are using a social strategy that is also more time-consuming (Thakkar et al., 2009). However, it should be noted here that other studies have shown better perspective taking to be linked to faster reaction times (e.g., Mohr et al., 2010; Thakkar and Park, 2010). Given that the above studies assessed empathy via self-report and that such approaches are problematic, we propose that future studies would benefit from using more objective measures of empathy (e.g., actual behavioral observations and/or facial mimicry) to examine whether higher empathy is associated with slower or faster reaction times. Another potential explanation of the above finding is that OT administration generally slows men down. It should be noted that whilst OT administration has been shown to slow men down in contexts other than spatial processing, such as during approach-avoidance motor responses to emotional faces (Theodoridou et al., 2013) and identification of fearful faces (Di Simplicio et al., 2009), it has not been found to have a slowing effect on approach-avoidance motor responses to non-social stimuli (Theodoridou et al., 2013), early processing (i.e., detection speed) of angry, and happy faces (Guastella et al., 2009a) and recognition of emotional faces (Fischer-Shofty et al., 2010). Therefore, it seems unlikely that OT administration generally slows men's speed of response. Importantly, the sex differences and the interaction with drug group were observed in a task that was introduced as a task that assesses empathy. Future research could introduce the task as one that assesses mental rotation (see also Massa et al., 2005 for instruction effects), to test whether drug effects are influenced by such context effects. This would provide us with some further insight regarding task expectancies, and their interaction with drug treatment. In any case, we suggest that the 3PP-task was powerful at showing drug by sex interactions because such reaction time measures are difficult to manipulate/see through relative to a self-report task, thereby rendering our participants less able to guess our specific experimental hypotheses. This advantage might also explain why no drug effect or drug by sex interaction was observed in study 1, in which participants are actively asked to rate their feelings toward an unfortunate person in the presence of the experimenter. We suggest that response biases such as social desirability and stereotyping come strongly into play when using such paradigms, resulting in artificially elevated scores and commonly observed sex differences (women having higher scores than men) when using self-report measures (see for example Massa et al., 2005; Wraga et al., 2007). Furthermore, this is unlikely to be the case when using implicit measures such as the current perspective taking task (Mohr et al., 2010, in press; Thakkar and Park, 2010; Gardner et al., 2012). Thus, our findings indicate that more implicit measures such as the current 3PP-task might be better suited to assessing empathy, and that any effects of empathy-related processes whether cognitive or neurochemical (such as OT in the present case) might emerge more consistently when using measures that are less response bias prone. 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: Collection of vaccination data in the German National CohortFindings of a feasibility study in three study centersImpfdatenerhebung in der Nationalen KohorteErgebnisse einer Machbarkeitsstudie in drei Studienzentren ABSTRACT.BACKGROUND: Data about the vaccination status of participants are required in epidemiological cohort studies whenever infection or immunity is considered as potential exposure or outcome. Within Pretest 2 of the German National Cohort (GNC) we therefore investigated the acceptance and feasibility of extracting vaccination status from vaccination certificates provided by the participants of the study. ABSTRACT.METHODS: This study was conducted in three study centers (Bremen, n = 73; Hamburg, n = 200; Hannover, n = 193). In order to test if an additional reminder would prevent participants from forgetting their vaccination certificates at home persons willing to participate in Pretest 2 were randomly assigned to one of three invitation groups (IG). About one third of the participants received either no further reminder (IG1), a reminder card together with the appointment letter (IG2) or a separate reminder card 4 days before the appointment (IG3). At the study center, vaccination data were scanned or copied and entered into a database using a unique identification number. Participants were also asked to fill in a short questionnaire to assess the completeness of the provided vaccination data. Additionally, in one of the three participating study centers, general practitioners (GP) were asked to provide vaccination data from their records following respective participants’ consent. Finally, we compared the influenza data from the vaccination certificates with the influenza data obtained from participants in Pretest 2 by use of a self-administered questionnaire (ID-Screen). ABSTRACT.RESULTS: Due to different starting dates of the study the intended reminder procedure was implemented only in Hamburg and Hannover. In Hamburg, significantly more vaccination certificates were submitted by the group which received the reminder card separately 4 days before the examination (IG3) compared to IG1 and IG2 (p = 0.04). In Hannover, in contrast, most vaccination certificates were brought by those who received the reminder card together with the appointment letter. Overall, the use of a reminder card had a positive but not significant effect as 89 % (185/209) of participants who received the reminder card submitted vaccination data versus 81 % (84/104) of participants who did not receive any reminder card (p = 0.06). Of all Pretest 2 participants in Hannover, 62 % (120/193) gave written consent for data collection by the GPs. In total, 114 practices were contacted of which 49 (43 %) sent vaccination data. All in all, 360 vaccination certificates with 5065 documented vaccinations were entered into a database, of which 4830 (95 %) were valid for analysis covering a period from 1946 to 2012. The comparison of influenza vaccination data from vaccination certificates to the remembered data from a self-completed questionnaire showed an agreement of data in 46 % (84/184) of cases (Kappa = 0.48). Influenza vaccinations were underreported in 4 % (7/170) of self-completed questionnaires. ABSTRACT.CONCLUSION: The reliable documentation of vaccinations within the context of the GNC proved to be feasible and thus recommendable at a large scale within the GNC as participants showed high willingness and compliance in providing available vaccination documents. An additional validation by means of documents provided by physicians seems to be possible for more than a quarter of participants. In order to maximize the likelyhood of participants’ of bringing their vaccination certificates it would be sufficient to send a reminder card together with the appointment letter. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi: 10.1007/s00103-014-2050-0) contains supplementary material, which is available to authorized users. For research questions in the field of infectious diseases and immunology within epidemiologic studies, data about the vaccination status of the participants are essential. Nevertheless evidence about immunity acquired by vaccinations at the population level is lacking [1]. On account of this insufficient data, research questions like the following have not been investigated so far: (a) vaccination coverage, specifically in adult age [2], (b) vaccine response and immunosenescence [3, 4] and (c) possible interactions between vaccinations, infections and chronic diseases [5, 6]. The German National Cohort (GNC) offers the opportunity to assess vaccination status in combination with the seroprevalences of a large population-based sample throughout Germany and to follow it up over many decades. Other than originally planned, the assessment of vaccinations is not part of the routine data collection of the GNC. Therefore, we conducted an add-on “Level 3” study in Pretest 2 to test the feasibility of vaccination data collection within the GNC but without interfering with the program of the GNC (for details see Ahrens et al. in this issue). Special focus was placed on (1) the acceptance and feasibility of vaccination certificate collection and means of response optimization, (2) the feasibility and benefit of additional vaccination data collection from GPs and (3) the comparison of vaccination data of different sources (vaccination certificates, data from GPs, self-reported influenza vaccinations) regarding completeness and validity. BODY.METHODS: BODY.RECRUITMENT: The study was conducted at three sites (Bremen, Hamburg, Hannover). Recruitment followed the standard operating procedure (SOP) of the GNC (Ahrens et al. in this issue). Accordingly, all persons who agreed to participate in Pretest 2 received a standardized invitation letter to confirm the appointment at the study center and to provide further information on how to prepare for the assessment. In this appointment letter participants were also asked to bring their vaccination certificates the day of the examination. To test whether an additional reminder would support participants to think of bringing their vaccination certificates, the use of a colored reminder card was tested (Fig. 1). For this purpose all participants were randomly assigned to one of three different invitation groups (IG). Participants of IG1 received the appointment letter only and no further reminder. Participants of IG2 received a colored reminder card together with the appointment letter, and participants of IG3 were sent this separate, colored reminder card 4 days before the appointment.Fig. 1Reminder card BODY.PROCEDURE OF VACCINATION DATA COLLECTION: At the study site, participants gave written informed consent. The vaccination data were scanned electronically (Hamburg, Hannover) or copied (Bremen) under a subject identification number. Personal data like name, address or date of birth were covered before scanning or copying the vaccination certificates. In case participants forgot to bring their vaccination certificate, they were instructed on how to send a copy of the vaccination certificate by mail. In order to assess the feasibility and benefit of additional vaccination data collection from GPs, participants in Hannover were also asked to consent to vaccination data collection from their GPs. If consent was obtained, the practices were contacted by mail. The letter included information about the Pretest 2 of the GNC, participants’ written consent and a documentation sheet for given vaccinations during the last 10 years which could be used in case that data could not be printed out. Finally, all retrieved vaccination data were entered into a database including date of vaccination, name of vaccine, batch number and (combination of) disease(s) to be prevented. If during the data entry questions arose which could not be solved, the entry was to be confirmed as “unfinished” and the reason put into a special field for notes. These unfinished cases were checked and completed as much as possible every other day. New vaccines not yet predefined in the database were added (dynamic list of vaccines). If the date of vaccination (day and/or month) was partly missing, illegible or cut off, the following approach was chosen for coding: the missing day was recorded as 15th of month, the missing month as “June” by keeping the information of recoding in order to be able to differentiate between the real vaccination date and a fictitious date. During the training phase of the personnel (first 50 vaccination certificates) every data entry was checked for quality of data by referring to the copies of vaccination certificates. Afterwards, additional quality checks were carried out for at least 40 % of entered vaccination certificates. BODY.QUESTIONNAIRE: A short questionnaire was filled in by participants during their stay at the study center to assess the completeness of the provided data from vaccination certificates. Questions were as follows: if the participant ever possessed a vaccination certificate, if at the time of examination at the study center a vaccination certificate was possessed, the number of vaccination certificates, if other vaccination documents exist, if all received vaccinations are recorded in the presented vaccination certificate and which vaccinations were performed but not documented in the vaccination certificates. One question was related to the attitude towards vaccinations as persons with a critical attitude may be reluctant to provide their vaccination certificate. Every mentioned missing vaccination was coded separately, so if influenza vaccinations of several years were indicated as missing, each vaccination was coded as one missing vaccination. Specific vaccinations mentioned as received during childhood were coded as reported (tetanus as tetanus) otherwise, if not further specified, summarized as “vaccinations received in childhood”. BODY.ETHICS APPROVAL: The study was approved by the Ethics Committee of the State Board of Physicians of the German Federal State of Lower Saxony (Ethikkommission der Ärztekammer Niedersachsen) and the federal State of Hamburg. BODY.RESULTS: BODY.PARTICIPATION: The recruitment process for this feasibility study differed among the participating study centers. On the one hand, the invitation procedure consisting of three different invitation groups was implemented in Hamburg and Hannover only, due to a later starting date in Bremen. Consequently, all participants in Bremen invited to Pretest 2 received by default the reminder card together with the appointment letter. On the other hand, participation proportions for this feasibility study could be calculated for Hannover only, as in Bremen and Hamburg the consent for the vaccination data collection was integrated into the consent form of the Pretest 2 baseline assessment whereas in Hannover participants were asked for a separate consent. Recruitment data and participation proportions per invitation group are shown in Fig. 2. The participation proportions of the random samples are of special interest because the study protocol for the GNC allows random samples only; therefore, participation proportions will be discussed mainly in this respect. Overall, 400 persons were invited to participate in this feasibility study. In Hannover, 92.2 % (153/166) of Pretest 2 participants gave a separate written consent for the vaccination data collection. Again 92.2 % (141/153) of these participants brought vaccination certificates to the appointment. In Hamburg and Bremen, where participants were not explicitly asked for their willingness to participate in this feasibility study, the proportion of provided vaccination certificates was slightly lower: 80.0 % (128/160, one missing) of invited persons in Hamburg and 75.3 % (55/73) in Bremen provided vaccination data. Fig. 2Flow chart of the recruitment and submitted vaccination certificates. RS = random sample from local registry, CS = convenience sample, consisting of volunteers, e.g. students, friends, colleagues and family members. Invitation group (IG): Appointment letter + … IG1: no further reminder; IG2: a colored reminder card together with the appointment letter; IG3: 4 days before the appointment at the study center a separate, colored reminder card (Fig. 1). *Hamburg: 1 missing case with no information about assigned invitation group, **Participation: Participants were asked for a separate consent for the vaccination data collection in Hannover only, ***Bremen: No documentation sheet about the invitation process available. 75 % of invited persons provided vaccination data the same day of examination or later. Considering the overall effect of the reminder card in Hamburg and Hannover, 89 % (185/209) of participants who received a reminder card (regardless if IG2 or IG3) brought their vaccination certificate compared to 81 % (84/104) of those who did not receive any reminder card (IG1; p = 0.06). The approach to send a separate reminder card 4 days before the appointment was the most successful; 92 % (94/102) participants of IG3 provided vaccination data compared to 85 % (91/107) in IG2 (p = 0.06). Stratifications by study center indicated that in Hannover participants in IG2 provided the most vaccination certificates (95 %), whereas in Hamburg, significantly more vaccination certificates (92 %) were provided in IG3, with no differences between IG1 and IG2 (chi-square2: 6.546, df 2, p = 0.04). BODY.DATA PROVIDED BY PHYSICIANS: Of 193 Pretest 2 participants in Hannover, 120 (62 %) provided written consent for the data collection by general practitioners. In one case the consent was given but the participant had not been in contact with the health care system for a long time so that he could not provide any GP address. In total, 114 practices were contacted of which 43 % (49/114) sent vaccination data. Two practices did not send any data because no vaccination was given during the past 10 years. In most cases the medical staff filled in the paper documentation sheet, with a few providing computerized print-outs. BODY.QUESTIONNAIRE: The short questionnaire was completed by 94.7 % (426/450) of participants including the convenience sample. There were considerable differences between the study centers. Whereas in Hamburg all participants completed the questionnaire (200/200), this was the case for 98.9 % (175/177) of the participants in Hannover, and for only 70 % (51/73) in Bremen (data shown in supplement). Thorough data cleaning was necessary because of inconsistent responses. For example, the question, if the participant ever possessed a vaccination certificate was denied by 16 persons of whom 5 persons submitted their vaccination certificate to the study personnel the same day. A problem seemed to exist also if more than one vaccination certificate existed so that participants were not sure to which one to refer. Moreover the high percentage of missing answers for most questions, coded as “don’t know/no answer”, suggests that these questions were difficult to remember and thus not suitable for a self-administered questionnaire. Of all participants, 96 % had possessed a vaccination certificate once during their life-time, with 91 % possessing one at the moment. There were no differences between random and convenience samples. In all, 37 % of responders indicated that the present vaccination certificate was the first, whereas for 55 % of responders it was the second up to fourth vaccination certificate. In addition, 67 % answered to have brought all available vaccination data to the study center, 54 % included all vaccinations ever received (random sample). Tetanus (18 %), influenza (17 %) and vaccinations received in childhood (13 %) were indicated most often as vaccinations not included in documentation. The question regarding the attitude towards vaccinations was answered by 416/426 (98 %) participants. The attitude towards vaccinations was assessed by 74 % of responders as positive. Of those 18 % being critical, 45 % indicated fear the side effects of vaccinations and 30 % were concerned that vaccinations might overwhelm the immune system. Among other reasons, responders took the chance to explain more in detail their critical attitude against specific vaccinations like influenza vaccination because of bad experiences. Only one person indicated to be in principle opposed to vaccinations; for 8 % of responders vaccinations were no issues of concern. No statistical difference of attitude towards vaccination could be detected between persons who submitted their vaccination certificate compared to those who did not submit any vaccination data. BODY.CAPTURING AND AVAILABILITY OF VACCINATION DATA: Overall 372 vaccination certificates were electronically captured or copied the same day of examination, of which 7 % (27/372) of vaccination certificates were lost primarily because of missing identification numbers (ID) on the copy/scan. The procedure of blackening the personal data before capturing the vaccination data electronically was not followed consequently so that in about 17 % of documents this needed to be worked over subsequently. The quality of vaccination certificates was partly poor, especially the older ones (before 1990) often lacked the names of given vaccines (1700 vaccines), or the date of vaccination. In 5 % (222) of cases, the vaccination date was missing, (partly) illegible or cut off during the process of scanning/copying the vaccination data. Some of these data (121 dates) could be recorded as described above. Further problems encountered were the lack of information about vaccines as single or combined vaccination or the illegibility of documented vaccinations. The median time for data entry was 14.3 min per vaccination certificate (minimum of 0.4 min; maximum of 124.3 min). Including the data of physicians, vaccination data could be obtained for a total of 82 % (367/450) of participants or 79 % (367/466) of all eligible Pretest 2 participants. An overview of submitted vaccination data per study center (number of valid vaccination documents and vaccinations) including the sample characteristics of persons submitting vaccination data is shown in Table 1. The documented vaccinations covered a period from 1946 to 2012. Table 1Submitted vaccination data and sample characteristics HamburgHannoverBremenTotalNo. of valid documents 158 155 (162a) 47 360 (367a) No. of vaccinations 2402 1968 (2186b) 510 4880 (5098b) No. of vaccinations/person 15.2 13.9 10.9 14.1 Mean (95 % CI) (13.8–16.6) (12.5–15.3) (8.7–13.0) (13.2–15.0) Sample characteristics n % n % n % n % Female 90 57.0 87 53.7 25 53.2 202 55.0 Male 68 43.0 75 46.3 22 46.8 165 45.0 20–29 years 23 14.6 18 11.1 3 6.4 44 12.0 30–39 years 15 9.5 17 10.5 - - 32 8.7 40–49 years 38 24.1 47 29.0 8 17.0 93 25.3 50–59 years 36 22.8 36 22.2 18 38.3 90 24.5 60–69 years 46 29.1 44 27.2 18 38.3 108 29.4 Born abroad 16 10.1 15 9.8 7 14.9 38 10.6 First language German 148 93.7 142 92.8 43 91.5 333 93.0 Married 59 37.3 88 57.5 23 48.9 170 47.5 Single 66 41.8 44 28.8 14 29.8 124 34.6 Full time employed 75 47.5 73 47.7 28 59.6 176 49.2 Monthly household net income (median): 2250 € up to  3000 € 83/146 56.8 73/141 51.8 19/46 41.3 175/333 52.6 N 158 162 47 367 GNC German National Cohort, GP general practitioners, DEGS Studie zur Gesundheit Erwachsener in Deutschland, GEDA Gesundheit in Deutschland aktuell, CI confidence interval a(…): Including data of seven participants in Hannover (4 of random and 3 of convenience sample) who provided physicians’ data only (with 33 documented vaccinations) as no vaccination certificates were available b(…): Including all vaccinations documented by physicians (n = 218) BODY.COMPARISON OF VACCINATION DATA PROVIDED BY PARTICIPANTS AND PHYSICIANS: For 40 participants in Hannover vaccination data from vaccination certificates and physicians’ documentation were available with a total of 630 documented vaccinations covering the period from 1956 to 2013. Most frequently documented vaccinations are shown in Table 2. Single vaccinations against poliomyelitis and tetanus were significantly more often, influenza vaccinations significantly less often documented in vaccination certificates than at the physicians’ offices. To assess the quality of vaccination documentation, the data captured by vaccination certificates were compared to data provided by physicians. Using the example of tetanus, 27 persons provided tetanus vaccination information documented in vaccination certificates (125 vaccinations) as well as documented by physicians (54 vaccinations). In all, 30 % (38/125) of tetanus vaccinations documented in the vaccination certificates corresponded exactly with tetanus vaccinations documented by physicians for 20/27 persons (74 %). In these cases the date of vaccination, the kind of vaccine and the producer of the vaccine were documented identically in each document. A total of 6 % (7/125) of documented tetanus vaccinations in the vaccination certificates did not correspond to documented tetanus vaccinations by physicians for 6/27 persons (22 %). Tetanus documentation differed because either the name of the vaccine was missing, not consistent or different producers of vaccines were indicated. In addition, 8/54 (15 %) tetanus vaccinations documented by physicians were not documented in the vaccination certificates which accounts for at least 6 % (8/125) of tetanus vaccinations missing in the vaccination certificates. Table 2Frequency of vaccinations documented in vaccination certificates compared to vaccinations documented by physicians Document byParticipant(1956–2012)a Physician(1964–2013)a Single poliomyelitis 88 20 % 13 7 % Single tetanus 68 15 % 14 8 % Tetanus (combined vaccine) 118 27 % 40 22 % Influenza 33 7 % 66 36 % Single hepatitis B 22 5 % 8 4 %21 Hepatitis A + B 20 5 % 14 8 % No. of vaccinations 446 184 Base: 40 participants with vaccination certificates and vaccination documentation from physicians aThe vaccination certificates covered a period from 1956–2012, the vaccination data sent by physicians from 1964–2013 BODY.VACCINATION STATUS: The vaccination coverage of responders (last dose no longer than 10 years ago) for some preventable diseases is shown in table 3. Table 3Vaccination coverage according to vaccination certificates HamburgHannoverBremenTotaln%n%n%n% (95 % CI)Tetanus Female 74 82 66 76 21 84 161 80 (59–74) Male 45 66 49 65 16 73 110 66 (59–74) Total 119 75 115 71 37 79 271 74 (69–78) Diphtheria Female 69 77 59 68 20 80 148 74 (67–80) Male 43 63 46 61 16 73 105 63 (56–71) Total 112 71 105 65 36 77 253 69 (64–74) Polio Female 50 56 39 45 13 52 102 51 (44–58) Male 26 38 30 40 12 55 68 41 (33–49) Total 76 48 69 43 25 53 170 46 (41–51) Hepatitis B Female 32 36 23 26 3 12 58 29 (23–35) Male 17 25 16 21 2 9 35 21 (15–27) Total 49 31 39 24 5 11 93 25 (21–30) Hepatitis A Female 28 32 23 26 5 20 56 28 (22–34) Male 23 33 16 21 1 5 40 24 (18–31) Total 51 32 39 24 6 13 96 26 (22–31) N 158 162 47 367 Measlesa Female 19 79 12 63 - - 31 72 (58–86) Male 12 86 11 69 2 67 25 76 (60–91) Total 31 82 23 66 2 67 56 74 (64–84) N 38 35 3 76 Number of persons who received at least one respective vaccination within the last 10 years aVaccination against measles in Germany recommended since 1974. Base: all participants of the age 20–39 years in the respective study centers who submitted their vaccination certificates BODY.COMPARISON OF DATA FROM A SELF-COMPLETED QUESTIONNAIRE (ID-SCREEN) VERSUS VACCINATION DATA FROM VACCINATION CERTIFICATES: In Pretest 2, the ID-Screen questionnaire, a self-administered questionnaire about infectious diseases, was applied in Hamburg and Hannover (see Castell et al. in this issue). Three questions about influenza vaccination were included: whether an influenza vaccination has ever been received (V1), if yes, in which frequency (V2) and the year of the last influenza vaccination (V3). In total 365 persons answered the influenza questions. Vaccination documents were available for 78 % (284/365) of these participants, which allowed assessing the agreement of self-reported versus documented influenza vaccinations. Half of all responders (184/365) indicated having ever been vaccinated against the flu (Table 4). Agreement with documentations was found for 84 (46 %) of these responders [Kappa 0.48] with up to 20 influenza vaccinations per person. Vaccination was underreported in 4 % (7/170) of cases where the receipt of influenza vaccination was negated but documented in the submitted vaccination records with up to 3 vaccinations per person. The “unvaccinated” responders were mostly vaccinated in 2008–2010 (7/11 vaccinations). The self-reported year of last influenza vaccination was confirmed by vaccination records in 27 % of cases (41/150). In 23 % (35/150) of cases the self-reported year did not correspond to the vaccination records because the year of the last remembered influenza vaccination was before (19 %) or after (5 %) the last influenza vaccination documented in the vaccination records. Table 4Comparison of influenza data from a self-completed questionnaire (ID- Screen) versus influenza vaccinations documented in vaccination records V1: ever vaccinatedV1: confirmedV2: every yearV3: last year confirmedn%n%n%n%Female 103 52.8 53 51.5 29 28.2 26/87 29.9 Male 81 47.6 31 38.3 27 33.3 15/63 23.8 Total 184/365 50.4 84 45.7 56 30.4 41/150 27.3 60–69 years 73/102 71.6 35 47.9 30 41.1 13/63 20.6 Hamburg 95/200 47.5 39 41.1 31 32.6 17/76 22.4 Hannovera 89/165 53.9 45 50.6 25 28.1 24/74 32.4 V1: ever vaccinated Have you ever been vaccinated against influenza? – yes (ID-Screen questionnaire) V1, confirmed Agreement between reported influenza vaccination (V1) and documented influenza vaccinations in vaccination certificates. V2 Self-reported regularity of influenza vaccinations in ID- Screen questionnaire (every year, almost every year, less often than every year, only once)V3, last year confirmed Agreement between self-reported year of last received influenza vaccination (n = 150) and vaccination date found in vaccination certificates aHannover only: Including vaccination data provided by physicians BODY.DISCUSSION: Vaccination data collection in Pretest 2 was shown to be feasible as participants were willing to provide vaccination documents. Documentation of vaccination for participants within the GNC would therefore be possible. Considering the completeness and quality of vaccination data it must be said that although the response to this feasibility study was good and much data could be retrieved, there are several limitations to consider. The documented vaccinations covered a period from 1946 to 2012 during which more and more vaccines became available and more vaccination recommendations were given. Not every administered vaccination is documented as vaccination certificates may be forgotten the day of vaccination. Also the fact that vaccinations are administered by different authorities (public health office especially before 1970, different physicians, company physicians, hospitals) accounts for missed vaccination documentation. Some data, like vaccinations given in childhood, are more likely to be missing especially if a person owns already the second or third vaccination certificate. As the comparison of vaccination certificates with data from physicians showed, which could be carried out for about a quarter of participants in Hannover, other received vaccinations may also be missing; however, it is difficult to assess to which extent. Some vaccinations could be found to be more often documented at the physicians’ but not in the vaccination certificate like influenza vaccinations. Also the comparison of self-reported influenza vaccination (ID-Screen, see Castell et al. in this issue) with the documented influenza data from certificates and physicians was limited by missing vaccination documentation which made it impossible to calculate the extent of overreporting. Underreporting of influenza vaccination occurred in at least 4 % of cases for whom vaccination documents were available. These problems are well-known and discussed in literature. Several studies have shown that neither self-reported vaccinations nor vaccination data based on medical records can be regarded as the gold standard for vaccination documentation [7, 8]. Miles et al. [9] concluded that vaccination cards tend to under-, self-reported vaccinations to overestimate coverage whereas a combination of both overestimated coverage in some studies and underestimated coverage in others ([9], S1565). Rolnick, who described underlying demographic factors for over- and under-reporting, recommends to improve not only vaccination documentation in medical records but also to improve the awareness of patients in order to get appropriate vaccination information ([7], S3933). As this feasibility study was an add-on within Pretest 2 and was not supposed to interfere with the assessment program of the GNC nor to use much of personnel or other resources there was no possibility for a personal-assisted interview to probe further for missing vaccination data. Despite the data gaps, we found the vaccination coverage of participants to be comparable to figures of other representative long-term studies such as DEGS, where the vaccination data were obtained from vaccination cards and/ or interviews or GEDA, a telephone survey. For example 74 % of Pretest 2 participants had sufficient tetanus vaccination coverage, compared to 71 % in DEGS1 [2] or 72 % in GEDA [10]. The incompleteness of vaccination data extracted from vaccination certificates should be put into perspective to the most important immunological questions addressed within the GNC (introduction), for which at least the vaccinations received during childhood and thus decades ago may be less important. Given the fact that for most vaccinations (except for Hepatitis B) no biomarkers are available to prove that vaccinations were carried out and at the same time the ability of participants to remember all ever received vaccinations is limited and probably also influenced by demographic factors [7], vaccination certificates seem to be at present the best possible population-based data source. The vaccination data provided by physicians were despite the limitations a useful supplement to the vaccination data of participants. Considering the time and effort required, the data collection from GPs would be feasible and useful only for specific research questions (influenza, or persons without vaccination certificates). Another aspect of the feasibility of our study refers to the training of the personnel. The electronic capturing and pseudonymization of vaccination data were defined in a standard operating procedure. Nevertheless at least 3 % of all submitted vaccination certificates got lost because of missing identification number. Other problems concerned poor quality of copies, incomplete data due to copying or scanning the data and failure to blacken the personal data dependably. All of these problems can be avoided by further training of study personnel. Also, well-trained staff is needed for the time-consuming data entry of vaccination data into the database. The use of a reminder card in addition to the standardized appointment letter in which participants were reminded to bring their vaccination certificate to the appointment had a positive effect on the proportion of provided vaccination certificates. If the reminder card should be sent in combination with the appointment letter or separately shortly before the visit in the study center could not be ascertained clearly as results for Hamburg and Hannover were inconsistent. The invitation procedure, especially the delay between appointment arrangements and sending of appointment letters and reminder card was difficult to control and might have varied among the participating study centers. Appointments were mostly made by phone and sometimes short-term, or cancelled and rearranged on short notice which made it often difficult to follow the routine for each invitation group. However, as the reminder card seemed to support participants to think of their vaccination certificates, we suggest sending the appointment letter together with the reminder card, which is not much additional effort. The short questionnaire which was intended to assess the completeness of provided vaccination data proved to be not suitable as a self-administered questionnaire. As the study protocol for the GNC is already extensive, a staff-assisted interview for these questions will not be feasible. The short questionnaire is therefore dispensable. Nevertheless the question referring to the attitude towards vaccinations was useful, as no statistical difference could be detected between those participants providing vaccination certificates and those who did not, meaning that participants who provided vaccination data were not less critical than persons who did not provide any vaccination data. No bias in this regard could be detected. Reasons why vaccination certificates were not provided may be the lack of documents but also the feeling that providing the vaccination certificate would be too personal. BODY.CONCLUSION: We demonstrated the feasibility of collecting vaccination data from vaccination certificates in a population-based study and recommend implementing this approach at a large scale within the German National Cohort in order to allow long-term prospective research on infection and immunity. BODY.ELECTRONIC SUPPLEMENTARY MATERIAL: (PDF 233 kb)

TITLE: Does L-carnitine improve endothelial function in hemodialysis patients? ABSTRACT.BACKGROUND:: Atherosclerosis is the leading cause of death in hemodialysis patients. These patients are also very prone to L-carnitine deficiency due to kidney disease. In this clinical trial, we investigated the effect of oral L-carnitine on endothelial function of these patients. ABSTRACT.MATERIALS ANS METHODS:: We studied 31 adult chronic hemodialysis patients in our center and divided them into two groups. The first group (n = 20) received 1500 mg/dialysis interval (every other day) oral L-carnitine. The control group (n = 11) received placebo for one month. Ultrasonographic measurements of flow mediated dilation and carotid intima-media thickness were performed before and after one month of L-carnitine and placebo therapy. ABSTRACT.RESULTS:: This study showed that after one month of L-carnitine or placebo therapy there was no significant improvement in flow mediated dilation (p = 0.80 and p = 0.59, respectively) or decrease in carotid intima-media thickness (p = 0.12 and p = 0.50, respectively). ABSTRACT.CONCLUSIONS:: Our study revealed that one month of oral L-carnitine therapy did not improve endothelial function in hemodialysis patients. Long-term studies with large sample size using intravenous form and higher doses of the drug are required to clarify the questionable role of L-carnitine in hemodialysis patients. BODY.INTRODUCTION: Atherosclerosis advances in renal failure and develops early in the course of renal dysfunction. Therefore, the consequences of atherosclerosis especially cardiovascular events represent a major clinical problem in these patients.[1] Cardiac death accounts for approximately 40-50% of all deaths and is the leading cause of death in these patients. Death from cardiovascular causes is up to 20 times more common in uremic patients than in the general population with the risk being even higher than in patients with diabetes mellitus.[1] Endothelial dysfunction (ED) seems to be the underlying cause of all vascular diseases. Accumulating evidence suggests that chronic renal failure (CRF) is associated with impaired endothelial cell function. ED is recognized as a key process in acute and chronic renal failure as well as end stage renal disease (ESRD) of all causes.[2] ED and intima-media thickness (IMT) are predictors for the development and progression of atherosclerosis.[3] The central role of endothelium in the development of vascular disease has led to identification of new relevant methods to estimate endothelial function and injury. Flow-mediated dilatation (FMD) is the most widely used noninvasive ultrasound method to assess endothelial function.[4] Measurement of ultrasound-based FMD in the brachial artery is noninvasive and relatively repeatable and reproducible, reflects important biology, and is useful in serial studies of disease reversibility. Brachial artery endothelium-dependent dilatation is also significantly correlated with findings in the coronary circulation in the same patients.[56] L-carnitine is a naturally occurring hydrophilic amino acid derivative, produced endogenously in the kidneys and liver and derived from meat and dairy products in the diet. It plays an essential role in the transfer of long-chain fatty acids into the mitochondria for beta-oxidation.[7] L-carnitine and short-chain acylcarnitines (esters of L-carnitine), such as acetyl-L-carnitine, are excreted by the kidneys. Renal reabsorption of L-carnitine is normally very efficient. In fact, an estimated 95% is thought to be reabsorbed by the kidneys.[7] Patients with renal disease who undergo hemodialysis are at risk for secondary carnitine deficiency. L-carnitine and many of its precursors are removed from the circulation during hemodialysis. Impaired L-carnitine synthesis by the kidneys may also contribute to the potential for carnitine deficiency in patients with end-stage renal failure undergoing hemodialysis.[7] As Guarnieri et al. noted, carnitine supplementation has been approved by the US Food and Drug Administration not only for the treatment, but also for the prevention of carnitine depletion in dialysis patients.[8] Previous studies have shown L-carnitine and its propionate to improve endothelial responses in animal models. L-carnitine has also been found to prevent the progression of atherosclerotic lesions. Endogenous carnitine depletion and/or carnitine deficiency should thus be viewed as an additional risk factor in atherogenesis.[910] However, many of the previous studies were performed on small numbers of patients. They were not well-controlled and did not include patients with CRF, although atherosclerosis and its related complications are the leading cause of mortality in CRF. To the best of our knowledge, this is the first study to evaluate the role of oral L-carnitine supplementation in improving endothelial function. For this purpose, FMD and carotid IMT in CRF patients were measured and compared with the values in a control group of patients with CRF who received placebo. BODY.MATERIALS AND METHODS: In a randomized double-blind, placebo-controlled trial, patients were recruited from the maintenance HD (Hemodialysis) population at Shafa Hospital, and Javadolaemeh and Samenolaemeh centers (Kerman, Iran) in 2011. This study was approved by research and ethics board at Kerman University of Medical Sciences, Kerman, Iran. Informed written consents were also obtained from all subjects. The number of cases was computed by NCSS statistical software using paired t-test. From previous studies, mean FMD changes before and after the intervention were considered as 7.5 and 6, respectively. Standard deviation (SD) was estimated as 2.5. Type I error probability (α) of 0.05, β of 20%, and power of 80% were also considered. Number of subjects in each group was thus calculated as 24 patients. Patients had been on hemodialysis 3 times weekly and for a period of at least 3 months. We excluded patients with infection, inflammation, incomplete or irregular drug or placebo intake, or arteriovenous fistula (AV) fistula for dialysis in both upper extremities. Smokers and individuals who received any new vasodilator agent during the study were also excluded. During the study period, we followed the patients for effective dialysis as scheduled and for any changes in their prescribed medication. Predialysis FMD and carotid IMT measurement by ultrasound were performed by the same operator in the L-carnitine and placebo groups before starting therapy and after one month of therapy. The patients were randomized to receive L-carnitine therapy or placebo. We administered 1500 mg oral L-carnitine or placebo between dialysis sessions in 3 divided doses in the mornings and nights except for the morning of days they underwent dialysis. All adverse effects during the course of the study in both case and control groups were recorded. BODY.MEASUREMENT OF FLOW-MEDIATED VASODILATATION: Using the guidelines suggested by Corretti et al.,[11] ultrasound assessment of endothelial-dependent FMD of brachial artery was investigated one hour before starting scheduled dialysis. Briefly, the study was performed in a temperature-controlled room (22°C) with the subjects in a resting and supine position. Brachial artery diameter was imaged using a 7.5-Mhz linear array transducer ultrasound system (Siemens Company). The brachial artery was imaged at a location 3-7 cm above the antecubital crease. To create a flow stimulus in the brachial artery, a sphygmomanometer cuff was placed on the forearm and the cuff was inflated 50 mmHg above systolic pressure to occlude artery flow for 5 minutes. All measurements were made at the end of diastole. The FMD was expressed as the percentage of change in post-stimulus diameter of the artery compared to the baseline diameter. BODY.MEASUREMENT OF CAROTID IMT: Longitudinal ultrasonographic scans of the carotid artery were obtained on the day of brachial artery reactivity assessment by FMD. The scans included the evaluation of the right common carotid artery 1 cm proximal to the carotid bulb. In each examination, the same operator used different scanning angles to identify the greatest IMT, defined as the distance between the junction of the lumen and intima and that of the media and adventitia in the far wall of the carotid artery. Finally, 3 measurements of carotid IMT were obtained from the right carotid artery and were averaged to determine the mean carotid IMT. Carotid IMT was expressed in millimeters. BODY.RANDOMIZATION AND BLINDING: A person, not involved in the study, assigned codes to the study groups and randomly allocated the selected participants to receive oral placebo or L-carnitine (1500 mg/day). The randomization was carried out by a procedure based on a random numeric sequence. The authors were unaware of treatment allocation. BODY.STATISTICAL METHODS: All analyses were carried out using SPSS13.0 (SPSS Inc., Chicago, IL, US). Categorical variables are reported as counts (percentage) and continuous variables as means ± SD unless otherwise indicated. To investigate the parameters in each group before and after therapy, t-test was used and to compare the data between different groups, analysis of variance (ANOVA) was performed. P values less than 0.05 were considered statistically significant. BODY.RESULTS: The calculated sample size in each group was 24 patients. However, since this trial was interventional and there was the possibility of interrupting the course of the treatment by patients or due to medication side effects, we started the study with 60 adult chronic hemodialysis patients and divided them randomly into two equal groups of L-carnitine and placebo. Only 20 patients in the L-carnitine and 11 in the placebo group continued the medication for one month and the rest of the cases were excluded from the study. The compliance in carnitine group was higher than that in the placebo group. The most common reason for discontinuing the drugs was their side effects, such as nausea and vomiting that were interestingly much more common in the placebo group (25% vs. 8%). The placebo was made exactly with the same basic ingredients and in the shape of L-carnitine tablets without including the L-carnitine component. Almost all patients were under multi-drug therapy for different complications of CRF. Therefore, the second common reason of not completing the course of therapy was that they were just tired of taking too many tablets. Finally, the data was obtained from 20 patients in the L-carnitine and 11 patients in the placebo groups. Sex and age distribution were comparable in the L-carnitine and control groups (male/female ratios of 16/4 and 7/4, respectively; p = 0.281 and 48.4 ± 13.8 and 51.2 ± 15.0 years old, respectively; p = 0.595). The duration of hemodialysis in the L-carnitine group was insignificantly longer than the placebo group (6.18 ± 6.73 vs. 2.95 ± 2.28 years; p = 0.394). Antihypertensive agents, mostly angiotensin converting enzyme inhibitors, were used regularly by 60% of the patients in the L-carnitine group and 80% of the patients in the placebo group. At the beginning of the study, FMD values measured in the L-carnitine and placebo groups were 8.63 ± 13.12 and 4.97 ± 3.33, respectively (p = 0.634). As Table 1 shows, FMD and carotid IMT values at the beginning and one month after the treatment were comparable, i.e. there was no statistically significant improvement in FMD or carotid IMT after 1 month of therapy. The difference between the two groups in the measured carotid IMT values at the beginning of the study was borderline (p = 0.05). Mann-Whitney test on FMD changes after one month of L-carnitine or placebo therapy showed the changes in the L-carnitine and placebo groups to be -1.019 and -0.0195, respectively (p = 0.71). Table 1Flow mediated dilation (FMD) and carotid intima-media thickness (IMT) changes in L-carnitine and placebo groups, before and after treatment Diabetics constituted 25% and 63% of the L-carnitine and placebo groups, respectively. In the beginning of the trial, hemoglobin A1c (HbA1c) was measured in diabetic patients. Considering 7% or less as normal HbA1c levels, only 41% of the patients had well-controlled diabetes mellitus. The influences of L-carnitine therapy on FMD in diabetic patients and also in nondiabetic patients were evaluated separately. The changes were not statistically significant in either diabetic patients (p = 0.625) or in nondiabetics (p = 0.678). Although at the beginning of the study, the measured FMD in the diabetic patients was lower than nondiabetics, the difference was not statistically significant (4.15 ± 3.38 vs. 6.84 ± 4.29, p = 0.108). In addition, there was no statistically significant difference in response after the therapy. Likewise, the chronicity of hemodialysis did not affect FMD changes by L-carnitine, as it was studied in patients with L-carnitine treatment who were under dialysis for less than or equal to 2 years (9.93 ± 18.25 and 6.43 ± 4.48; p = 0.508), and those with more than two years of initiating dialysis (6.09 ± 3.79 and 6.77 ± 4.58; p = 0.815). While 25% of the patients in the L-carnitine group reported a sense of well being following taking the medication, no patient in the placebo group had such a feeling. Loffredo et al. measured FMD in 40 normal patients with a mean age of 64.2 ± 11.2 years. They indicated the mean percentage changes of FMD as 10.34 ± 2.14.[12] We compared it with the mean percentage changes of FMD measured in CRF patients (5.57 ± 4.58) at the beginning of our study (with removing just one value that seemed to be out of the normal range) and the difference was statistically significant (p < 0.005). In other words, FMD was impaired in CRF patients compared to the normal population. BODY.DISCUSSION: This study demonstrated that one month of oral L-carnitine supplementation was not associated with improvement in endothelial function as measured by FMD and carotid IMT in CRF patients on chronic hemodialysis. In fact, our data indicated that the disease had a progressive course and L-carnitine could not influence on it significantly. A number of studies have been conducted over the past years to assess the efficacy of supplemental L-carnitine on improving some clinical complaints of CRF patients. However, there has been no trial regarding the role of L-carnitine on the endothelial function in these patients. Considering the high prevalence of atherosclerosis and related complications in CRF patients, we conducted our study to investigate this concept. Stasi et al.[13] and McMackin et al.[14] demonstrated that propionyl-L-Carnitine accelerated blood flow recovery and the restoration of vascular function in animal models. Loffredo et al. showed that propionyl-L-carnitine infusion was associated with increased FMD in patients with peripheral vascular disease.[12] In another study, Volek et al. reported that L-carnitine supplementation improved FMD in healthy individuals after high fat meals (which has been shown to cause impairment of vascular health by reducing FMD).[15] Silvestro et al. concluded that in patients with intermittent claudication, propionyl-L-carnitine administration provided a protective effect against deterioration of FMD and increase of soluble vascular cell adhesion molecule-1 (sVCAM-1) induced by exercise.[16] Previous studies in animal models have shown that L-carnitine and its propionate improved endothelial responses by decreasing O2 production and increasing nitric oxide (NO) availability. They also suggested that L-carnitine prevents the progression of atherosclerotic lesions and endogenous carnitine depletion and/or deficiency should thus be viewed as an additional risk factor in atherogenesis.[910] There has been no previous study to investigate the role of L-carnitine therapy on improvement of endothelial function in CRF patients. In this clinical trial, we found that L-carnitine did not change FMD or carotid IMT in CRF patients after one month of therapy. Some explanations can justify this finding. Considering the risk of accumulation of toxic metabolites of L-carnitine such as trimethylamine (TMA) in CRF patients, we prescribed 1500 mg of oral L-carnitine supplementation between dialysis sessions and only for one month. However, the bioavailability of oral L-carnitine is about 15%.[1718] Though the recommended form of L-carnitine in ESRD is the intravenous form, unfortunately it does not exist in the country of study (Iran). Therefore, the oral form and the relatively short duration of prescription of L-carnitine might have influenced on our results. Further clinical trials with intravenous form of L-carnitine should be performed to increase not only the bioavailability of the drug, but also the compliance of the patients. There will be also no risk of toxic metabolites accumulation with prescribing the intravenous form. On the other hand, most CRF patients receive antihypertensive agents, especially angiotensin converting enzyme inhibitors that mediate NO-related vasodilatation and increase baseline vessel diameter which in turn leads to smaller estimates of FMD including the brachial artery FMD. This might have effects on the percentage of changes of FMD after therapy.[19] Moreover, since most of the time a chronic insidious disease such as diabetes mellitus leads to CRF, well-established atherosclerosis is usually encountered at the time of starting dialysis and this might have a marked influence on patients’ response to L-carnitine. Based on Stephens et al., carbohydrate rich foods, which induce a state of hyperinsulinemia, appear to improve carnitine transport into the skeletal muscles.[20] Bloomer et al. suggested that this may also be true as related to uptake into endothelial cells.[21] If approved, considering the fact that the most common cause of CRF in adults is diabetes mellitus, abnormal insulin secretion and resistance to insulin, may minimize the effect of L-carnitine in the patients. We conclude that the role of L-carnitine in chronic hemodialysis patients is still questionable. Our clinical trial demonstrated no significant improvement in endothelial function in these patients after one month of therapy. Additional randomized controlled clinical trials with larger sample sizes and sufficient power may lead to a better understanding of the possible beneficial effects of L-carnitine on hemodialysis patients.

TITLE: Hip arthroscopy versus best conservative care for the treatment of femoroacetabular impingement syndrome (UK FASHIoN): a multicentre randomised controlled trial ABSTRACT.SUMMARY: ABSTRACT.BACKGROUND: Femoroacetabular impingement syndrome is an important cause of hip pain in young adults. It can be treated by arthroscopic hip surgery, including reshaping the hip, or with physiotherapist-led conservative care. We aimed to compare the clinical effectiveness of hip arthroscopy with best conservative care. ABSTRACT.METHODS: UK FASHIoN is a pragmatic, multicentre, assessor-blinded randomised controlled trial, done at 23 National Health Service hospitals in the UK. We enrolled patients with femoroacetabular impingement syndrome who presented at these hospitals. Eligible patients were at least 16 years old, had hip pain with radiographic features of cam or pincer morphology but no osteoarthritis, and were believed to be likely to benefit from hip arthroscopy. Patients with bilateral femoroacetabular impingement syndrome were eligible; only the most symptomatic hip was randomly assigned to treatment and followed-up. Participants were randomly allocated (1:1) to receive hip arthroscopy or personalised hip therapy (an individualised, supervised, and progressive physiotherapist-led programme of conservative care). Randomisation was stratified by impingement type and recruiting centre and was done by research staff at each hospital, using a central telephone randomisation service. Patients and treating clinicians were not masked to treatment allocation, but researchers who collected the outcome assessments and analysed the results were masked. The primary outcome was hip-related quality of life, as measured by the patient-reported International Hip Outcome Tool (iHOT-33) 12 months after randomisation, and analysed in all eligible participants who were allocated to treatment (the intention-to-treat population). This trial is registered as an International Standard Randomised Controlled Trial, number ISRCTN64081839, and is closed to recruitment. ABSTRACT.FINDINGS: Between July 20, 2012, and July 15, 2016, we identified 648 eligible patients and recruited 348 participants: 171 participants were allocated to receive hip arthroscopy and 177 to receive personalised hip therapy. Three further patients were excluded from the trial after randomisation because they did not meet the eligibility criteria. Follow-up at the primary outcome assessment was 92% (319 of 348 participants). At 12 months after randomisation, mean iHOT-33 scores had improved from 39·2 (SD 20·9) to 58·8 (27·2) for participants in the hip arthroscopy group, and from 35·6 (18·2) to 49·7 (25·5) in the personalised hip therapy group. In the primary analysis, the mean difference in iHOT-33 scores, adjusted for impingement type, sex, baseline iHOT-33 score, and centre, was 6·8 (95% CI 1·7–12·0) in favour of hip arthroscopy (p=0·0093). This estimate of treatment effect exceeded the minimum clinically important difference (6·1 points). There were 147 patient-reported adverse events (in 100 [72%] of 138 patients) in the hip arthroscopy group) versus 102 events (in 88 [60%] of 146 patients) in the personalised hip therapy group, with muscle soreness being the most common of these (58 [42%] vs 69 [47%]). There were seven serious adverse events reported by participating hospitals. Five (83%) of six serious adverse events in the hip arthroscopy group were related to treatment, and the one in the personalised hip therapy group was not. There were no treatment-related deaths, but one patient in the hip arthroscopy group developed a hip joint infection after surgery. ABSTRACT.INTERPRETATION: Hip arthroscopy and personalised hip therapy both improved hip-related quality of life for patients with femoroacetabular impingement syndrome. Hip arthroscopy led to a greater improvement than did personalised hip therapy, and this difference was clinically significant. Further follow-up will reveal whether the clinical benefits of hip arthroscopy are maintained and whether it is cost effective in the long term. ABSTRACT.FUNDING: The Health Technology Assessment Programme of the National Institute of Health Research. BODY.INTRODUCTION: Femoroacetabular impingement syndrome is a painful disorder of the hip that is caused by a premature contact (impingement) between the femur and acetabulum during hip movements.1, 2 This premature contact typically occurs as a result of certain hip shapes, such as cam or pincer morphology.1 Cam morphology refers to a flattening or convexity at the femoral head neck junction, whereas pincer morphology refers to a focal or global over-coverage of the femoral head by the acetabulum.1 Femoroacetabular impingement syndrome leads to progressive damage within the joint, including the acetabular labrum and articular cartilage,1 and is associated with the development of osteoarthritis of the hip.1, 3 BODY.RESEARCH IN CONTEXT: Evidence before this study Femoroacetabular impingement syndrome is a relatively common non-arthritic cause of hip pain in young adults. It is caused by symptomatic premature contact between the proximal femur and acetabular rim during hip motion, and this phenomenon is associated with certain hip shapes. Repeated impingement leads to damage to the articular cartilage or to acetabular labral tears, and it is these injuries that are thought to be painful. In the past decade, surgeons have developed arthroscopic (ie, keyhole) surgical techniques to treat femoroacetabular impingement syndrome. The rationale is that reshaping the hip and repairing cartilage and labral damage will prevent impingement and relieve symptoms. In 2013, we did a Cochrane systematic review of the effectiveness of hip arthroscopy for femoroacetabular impingement syndrome. We searched MEDLINE (from 1946 to Nov 19, 2013), Embase (1980 to Nov 19, 2013), and the Cochrane Central Register of Controlled Trials (issue 11, 2013), for randomised controlled trials of surgery compared with placebo treatment, non-operative treatment, or no treatment, in any language, in human adults with femoroacetabular impingement syndrome. We found no high-quality evidence examining the effectiveness of surgery for femoroacetabular impingement syndrome. Therefore, we chose to do a pragmatic, multicentre, randomised controlled trial to determine the effectiveness of hip arthroscopy compared with best conservative care. Added value of this study To our knowledge, this is the largest randomised controlled trial to show the clinical effectiveness of hip arthroscopy in treating femoroacetabular impingement syndrome. We have shown that patients with femoroacetabular impingement syndrome have better hip-related quality of life at 12 months since randomisation with either hip arthroscopy or best conservative care (personalised hip therapy), and that this quality of life improves significantly more in patients treated hip arthroscopy, exceeding the minimal clinically important difference. Hip arthroscopy was also more expensive than was personalised hip therapy. Implications of all the available evidence Over the past 15 years, increasing numbers of patients with femoroacetabular impingement syndrome have been treated with hip arthroscopy. This is the first study to show that hip arthroscopy is more clinically effective, at least in the short term, than best conservative care. Longer-term outcomes are required to establish whether this improvement is sustained and whether surgery is cost-effective. These results should be shared with patients when selecting an appropriate treatment strategy. Surgery has become an established treatment for femoroacetabular impingement syndrome. The aim of such surgery is to reshape the hip joint to prevent impingement.2 Intra-articular injury, such as a cartilage and labral damage, can be resected, repaired, or reconstructed.2 Initially, open surgery was used to treat femoroacetabular impingement syndrome, but the minimally invasive, so-called keyhole technique known as hip arthroscopy is now being used more frequently.1 This approach has become possible because of advances in technology and surgical techniques. Hip arthroscopy is safer and has a shorter recovery time than does open surgery.4, 5 In the UK in 2013, 1908 operations for femoroacetabular impingement syndrome were done by arthroscopic surgery, compared with only 491 by open surgery.6 Since then, there has been a rapid increase in the use of hip arthroscopy in most countries around the world.4, 5, 6 Non-operative treatments for femoroacetabular impingement syndrome are also available,2 including exercise-based packages of conservative care, delivered by a physiotherapist.7 Potential targets for physiotherapy include the abnormal movement patterns and weakness of hip muscles seen in patients with femoroacetabular impingement syndrome.8, 9 Numerous case series report improvement in patients with femoroacetabular impingement syndrome after open or arthroscopic surgery, or physiotherapy.5, 10 However, a 2014 Cochrane review11 of surgery for treating femoroacetabular impingement syndrome showed that there was no evidence from randomised controlled trials to support these treatments. In a feasibility study,6 we established that patients were prepared to be recruited, and that surgeons were in equipoise and willing to recruit patients to a randomised controlled trial of hip arthroscopy compared with best conservative care. Our aim was to measure the clinical effectiveness of hip arthroscopy compared with best conservative care in treating patients with femoroacetabular impingement syndrome. BODY.METHODS: BODY.STUDY DESIGN AND PARTICIPANTS: We did this pragmatic, multicentre, assessor-blind randomised controlled trial in 23 National Health Service (NHS) hospitals in the UK. We treated the initial feasibility study6 as an internal pilot study so that participants who took part in the initial study were included in the main trial recruitment. Participants were recruited from the specialist hip arthroscopy service at each hospital. Participating surgeons identified eligible patients during routine diagnostic consultations. Assessments were medical history, clinical examination, X-rays, and cross-sectional imaging (MRI, CT, or both). For patients with a diagnosis of femoroacetabular impingement syndrome, the surgeon classified them as having cam (alpha angle >55°), pincer (lateral centre-edge angle >40° or a positive crossover sign), or mixed-type (combination of cam and pincer) impingement.12 Patients were eligible if they had hip pain, radiographic features of cam or pincer morphology, were at least 16 years old, were able to give informed consent, and if the treating surgeon believed that they were likely to benefit from hip arthroscopy. Patients were excluded if they had hip osteoarthritis (Tonnis grade >1 or less than 2 mm of superior joint space on an antero-posterior radiograph); a history of hip pathology such as Perthes' disease, slipped upper femoral epiphysis, or avascular necrosis, or previous hip injury such as acetabular fracture, hip dislocation, or femoral neck fracture; or if they had already had shape-changing surgery (open or arthroscopic) of the hip.13, 14 Patients with bilateral femoroacetabular impingement syndrome were eligible, and only the most symptomatic hip was randomly assigned to treatment and followed up. Trained research associates approached eligible patients to explain the trial and to invite them to participate. All participants gave written informed consent. Qualitative research, to understand recruitment as it occurred, was integrated into the trial. The findings were used to design a recruiter training and centre support programme that was implemented during the trial to optimise recruitment. The research was based on the QuinteT Recruitment Intervention and continued the work done during the internal pilot trial.15, 16 The study was approved by NHS Research Ethics Service West Midlands (14/WM/0124). An independent trial steering committee and data monitoring committee provided oversight of the progression of the study. The study protocol has previously been published.17 BODY.RANDOMISATION AND MASKING: Participants were randomly assigned (1:1) with a computer-generated minimisation (adaptive stratified sampling) algorithm for centre and type of impingement, to receive either hip arthroscopy or best conservative care. All baseline data were collected before randomisation, which was done by the recruiting research associate. Allocation concealment was ensured by use of a secure telephone randomisation service hosted by Warwick Clinical Trials Unit. It was not possible to mask patients or the treating clinicians to their allocation. Researchers who collected outcome assessments and analysed the results were masked to allocation by concealment of treatment. BODY.PROCEDURES: Surgery for femoroacetabular impingement syndrome was done using arthroscopic techniques by a senior surgeon (consultant grade) in the NHS, who was trained and experienced in hip arthroscopy. Further details about surgeons' training and experience, and surgical procedures are shown in the appendix. 27 trial surgeons did the surgery. Hip arthroscopy was done under general anaesthesia in a lateral or supine position. Arthroscopic portals were established in the central and peripheral compartments of the hip under radiographic guidance according to the surgeon's usual practice. Shape abnormalities and consequent labral and cartilage pathology were treated. Adequacy of bony reshaping was assessed by intraoperative image intensifier views or by arthroscopic visualisation of a satisfactory impingement free range of movement of the hip, or both. Patients were allowed home when they could walk safely with crutches (typically within 24 h). Patients were referred to outpatient physiotherapy services for a course of rehabilitation as per usual care for that surgeon. These post-operative physiotherapists were distinct from those providing conservative care, to avoid contamination between groups. Patients had MRI of their hip at least 6 weeks after surgery. A panel of international experts assessed the fidelity of the surgery (appendix). They reviewed operation notes, intraoperative images, and postoperative MRI scans to subjectively assess whether adequate surgery had been undertaken, according to the protocol. The panel discussed each case and subjectively assessed whether shape abnormalities and intra-articular pathology were treated, and whether there was a sufficient resection to allow impingement-free range of motion. This approach included assessing the proximal femoral and acetabular rim resections and whether the resection edges were smooth. Personalised hip therapy is a package of physiotherapist-led rehabilitation for femoroacetabular impingement syndrome. It was developed during our feasibility study and tested during the pilot study.6 Although the name for this intervention is new, the care offered was based on a consensus of what physiotherapists, physicians, and surgeons currently regard as best conservative care for femoroacetabular impingement syndrome.7 Personalised hip therapy has four core components: an assessment of pain, function, and range of hip motion; patient education; an exercise programme taught in the clinic and repeated at home, that has the key features of individualisation, progression, and supervision; and help with pain relief, which could include one X-ray or ultrasound-guided intra-articular steroid injection when pain prevents performance of the exercise programme.18 Personalised hip therapy was delivered by at least one physiotherapist at each centre. 47 physiotherapists were trained formally in this protocol through a 1-day workshop and supported to deliver personalised hip therapy through refresher workshops (appendix). At their initial assessment, participants received a personalised hip therapy information pack that described what to expect during the course of their treatment. They then had between six and ten face-to-face contacts with the physiotherapist over 12–24 weeks. Some contacts were allowed by either telephone or email when geographical distance prevented all contacts being carried out face-to-face. Patient-completed exercise diaries were also encouraged to help both patients and physiotherapists monitor progress and adherence with personalised hip therapy. Physiotherapists recorded full details of their advice and treatments, number and type of treatment contacts, and any non-attendance, on case report forms. These case report forms were reviewed for accuracy in comparison to the usual physiotherapy records at each treatment centre and then assessed for fidelity to the personalised hip therapy protocol by a panel comprising members of the core group who developed the protocol for personalised hip therapy (appendix). The personalised hip therapy panel reviewed case report forms to ensure participants received an adequate number of sessions, all four core components, and that their exercise programme was individualised, supervised, and progressive. BODY.OUTCOMES: The primary outcome was hip-related quality of life, as measured by the international Hip Outcome Tool (iHOT-33) at 12 months after randomisation.19 iHOT-33 is a patient-derived and patient-reported outcome instrument designed to measure hip-related quality of life in young adults with non-arthritic hip pain. The iHOT-33 consists of four domains: symptoms and functional limitations; sports and recreational physical activities; job-related concerns; and social, emotional, and lifestyle concerns.18 iHOT-33 provides a 100-point score, with 100 representing no pain and perfect function, and lower scores indicating pain and poor function. The instrument has been validated in a relevant population for this trial,19 and has a minimum clinically important difference of 6·1 points.19, 20 Secondary outcomes were health-related quality of life, measured with the EuroQol EQ-5D-5L, the 12-item Short Form Health Survey (SF-12 version 2), adverse events, and health-care resource use, all measured 6 and 12 months after treatment allocation.21, 22, 23 Patients reported adverse events 6 weeks after the start of their intervention (first session of personalised hip therapy, or date of hip arthroscopy). Any serious adverse events were reported by each centre to the Warwick Clinical Trials Unit. Forms for iHOT-33, EQ-5D-5L, SF-12, adverse events, and health-care resource use were collected by postal questionnaires, which were centrally administered. Scores for these measures were collected at the time of consent and again by postal questionnaire at 6 and 12 months after treatment allocation. BODY.STATISTICAL ANALYSIS: The planned sample size was 172 participants in each group, based on a standard deviation of iHOT-33 of 16 points and a minimum clinically important difference of 6·1 points, giving a standardised effect size of 0·38. We designed the trial to have 90% power to detect an effect of this size at a two-sided 5% significance level, allowing for up to 15% loss to follow-up at the primary outcome timepoint. We prepared a full statistical analysis plan before the final analysis. No interim analyses were planned. The plan was approved by the independent data monitoring committee. In our primary analysis, we investigated differences in the primary outcome measure, (iHOT-33 score at 12 months after randomisation) between the two treatment groups on a intention-to-treat basis, which included all eligible patients randomly assigned to an intervention. We assessed the primary outcome 12 months from treatment allocation rather than from intervention because this was a pragmatic trial design of two different treatment strategies. We used a mixed-effects regression analysis to assess the effects of the interventions on 12-month iHOT-33 scores, after adjusting for the fixed-effects of impingement type, sex, and baseline iHOT-33 score, with recruiting centre included as a random effect to model any potential associations within the recruiting centres. This mixed-effects model was used for all outcome measures. We drew our primary inferences from the intention-to-treat analysis, irrespective of compliance, without imputation for missing data. We also did several additional analyses. We did a per-protocol analysis, comparing participants who actually received surgery and those who actually received personalised hip therapy. Additionally, we compared participants randomly allocated to hip arthroscopy or personalised hip therapy, who received an allocated treatment that was deemed to be of high fidelity. We did prespecified subgroup analyses for different impingement types (cam, pincer, and mixed) and for patients younger and older than 40 years. We added pairwise interaction terms between treatment group and both impingement type and age group to the mixed-effects model to test for important subgroup effects. In addition to the primary adjusted analysis, we also reported unadjusted differences between groups, and we assessed significance using t tests for normally distributed outcomes. We present treatment effect estimates from the adjusted mixed-effects model (primary analysis) with 95% CIs. All hypothesis testing was at the 5% level, with no adjustments for multiple testing. Unless otherwise indicated, the analysis of secondary outcomes followed the same modelling approach as for the primary outcome. All analyses were on a complete-case basis and where follow-up data were missing, the reasons for missing data were obtained and patterns were investigated to judge the plausibility of missingness assumptions. We did a sensitivity analysis using multiple imputation techniques (imputation using chained equations) to assess the effect of missing data on the primary outcome. Finally, we did a post-hoc sensitivity analysis of the effect of variation in recovery time in the hip arthroscopy group. We compared post-intervention adverse events between groups using Fisher's exact test. We did all analyses using Stata (version 14).24 We did an economic evaluation from the perspective of the NHS and personal social services.25 We estimated economic costs associated with the delivery of the two interventions. Resource use questions completed by participants at each assessment point provided a profile of all hospital inpatient and outpatient service use, community health and social care encounters, prescribed medications, and NHS supplies, such as crutches or home adaptations. We obtained unit costs (2016 £) from primary and secondary sources in accordance with national guidelines and attached them to every item of resource use.24 We used health utilities generated from EQ-5D-5L responses at every timepoint of assessment to estimate quality-adjusted life-year (QALY) profiles for every participant; these QALYs were calculated as the area under the baseline-adjusted utility curve, assuming linear interpolation between utility measurements. We did a bivariate regression of costs and QALYs, with multiple imputation of missing data, with the view to estimating the incremental cost per QALY gained for hip arthroscopy compared with personalised hip therapy. Further details, including sensitivity analyses done to assess the effect of uncertainty surrounding aspects of the economic evaluation, and prespecified subgroup analyses exploring heterogeneity in the cost-effectiveness results, are provided in the appendix. The trial is registered as an International Standard Randomised Controlled Trial, number ISRCTN64081839. 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 final responsibility for the decision to submit for publication. BODY.RESULTS: Between July 20, 2012, and July 15, 2016, 648 patients who attended the participating surgeons' hip clinics were deemed eligible (figure 1). Of these patients, 348 (54%) agreed to participate. The mean age of participants was 35·3 years (SD 9·6); the mean age of those who declined to participate was 35·4 years (10·3). The 348 participants were randomly allocated to receive hip arthroscopy (n=171) or personalised hip therapy (n=177). Three additional patients were randomly allocated in error, in each instance contrary to protocol procedures. One participant, who was not eligible, was recruited after miscommunication between the treating surgeon and recruiter; one patient was allocated without signing the consent form; and one patient was deemed eligible by a trainee surgeon, but within a few days the treating surgeon deemed they were not eligible. None of these patients participated in the trial (figure 1).Figure 1Trial profile iHOT-33=International Hip Outcome Tool. *Three patients were randomly assigned in error but did not receive treatment and were not followed-up. Participants in the two groups were well matched in terms of demographics and pre-randomisation hip-related quality of life, having had symptoms for approximately 3 years (table 1). 14 (8%) participants who were allocated to personalised hip therapy had all or part of this intervention, but then, at their request, went on to have hip arthroscopy within 12 months after randomisation. No patients allocated to hip arthroscopy had personalised hip therapy. For hip arthroscopy, the median time from random assignment to treatment was 122 days (IQR 80–185), and for personalised hip therapy, it was 37 days (22–60). Surgeons did a mean of 112 (SD 55) hip arthroscopies per year during the study. At 12 months after randomisation, 144 (84%) of the 171 participants allocated to receive hip arthroscopy had received it; 27 (16%) had not. 121 (84%) participants who received a hip arthroscopy procedure had postoperative MRI and their case was assessed by the surgical review panel; 105 (87%) of these 121 procedures were deemed to be of high fidelity, and 16 (13%) were deemed unsatisfactory. The reasons for unsatisfactory surgery were an inadequate bony resection of the proximal femur (n=7) and acetabular rim (n=2), a sharp transition from the femoral head to neck as a result of reshaping surgery (n=5), and no reshaping surgery being done because the hip was degenerate (n=2). Nine (5%) of the 177 participants allocated to personalised hip therapy did not receive any treatment by 12 months. Of the patients who received personalised hip therapy (n=154), 107 (70%) were judged to have received the intervention to a high fidelity (appendix). The most common reason for low fidelity of personalised hip therapy was participants not receiving the minimum of six therapy sessions (34 [72%] of 47). Other reasons for low-fidelity personalised hip therapy were no progression of exercises by the physiotherapist (11 [23%] of 47) and the patient not complying with the exercise programme (two [4%] of 47).Table 1Baseline characteristics of the study population Hip arthroscopy (n=171)Personalised hip therapy (n=177)Age (years) 35·4 (9·7) 35·2 (9·4) Sex Women 71 (42%) 64 (36%) Men 100 (58%) 113 (64%) Current smoker Yes 31 (18%) 25 (14%) No 136 (80%) 151 (85%) Missing data 4 (2%) 1 (1%) Hip side considered for treatment Right 95 (56%) 103 (58%) Left 75 (44%) 74 (42%) Participants with bilateral symptoms 11 (6%) 18 (10%) Duration of hip symptoms (months) 37 (36·6) 40 (40·8) Impingement type Cam 129 (75%) 133 (75%) Mixed 29 (17%) 30 (17%) Pincer 13 (8%) 14 (8%) Units of alcohol in an average week 6·2 (8·6) 6·0 (7·7) Diabetes Yes 2 (1%) 4 (2%) No 165 (96%) 171 (97%) Missing data 4 (2%) 2 (1%) Chronic renal failure Yes 1 (1%) 0 No 166 (97%) 176 (99%) Missing data 4 (2%) 1 (1%) Physical activity (UCLA Activity Scale) 4·3 (2·5) 4·4 (2·5) Hip-related quality of life (iHOT-33) 39·2 (20·9) 35·6 (18·2) SF-12 PCS 44 (7·6) 44 (5·9) SF-12 MCS 42 (7·1) 42 (7·3) EQ-5D 3L/5L Index Score 0·576 (0·26) 0·557 (0·25) EQ-5D 5L VAS 67 (20·2) 67 (18·7) Mean lateral centre edge angle (°) 31 (5) 31 (5) Number of participants with LCEA <25° 7 (4%) 6 (3%) Number of participants with LCEA <20° 0 0 Mean alpha angle (°) measured on antero-posterior radiograph 61 (17) 64 (18) Data are mean (SD) or n (%). UCLA=University of California Los Angeles. iHOT-33=International Hip Outcome Tool. SF-12=12-item Short Form Health Survey. PCS=physical component score. MCS=mental component score. VAS=visual analogue score. LCEA=lateral centre edge angle. 319 (92%) of 348 participants completed the iHOT-33 questionnaires at 12 months after randomisation. Seven (2%) withdrew from follow-up, and 22 (6%) were lost to follow-up. The iHOT-33 score increased in both groups, indicating an improvement in hip-related quality of life (table 2, figure 2). 12 months after randomisation, mean iHOT-33 scores had improved from 39·2 (SD 21) to 58·8 (27) for participants in the hip arthroscopy group, and from 35·6 (18) to 49·7 (25) in the personalised hip therapy group. In the primary intention-to-treat analysis at 12 months, the adjusted estimate of treatment effect measured with iHOT-33 was 6·8 (95% CI 1·7 to 12·0, p=0·0093) in favour of hip arthroscopy, compared with personalised hip therapy. In the per-protocol analysis at 12 months, including participants who received personalised hip therapy (n=154) or hip arthroscopy (n=144), the adjusted estimate of the between-group difference on iHOT-33 was 8·2 (95% CI 2·8 to 13·6) in favour of hip arthroscopy. In the exploratory, pre-specified secondary analysis based on those participants whose treatment was deemed of a high fidelity (hip arthroscopy n=105, personalised hip therapy n=107), the adjusted estimate of between-group difference on the iHOT-33 was 5·8 (95% CI −0·7 to 12·2) in favour of hip arthroscopy. There were no significant between-group differences in SF-12 or EQ-5D-5L scores at 6 or 12 months after randomisation (table 2).Figure 2Changes in mean iHOT-33 score from baseline to 6 and 12 months after randomisation Error bars are 95% CIs. iHOT-33=International Hip Outcome Tool. Table 2Patient-reported outcome measures Hip arthroscopy (n=171)Personalised hip therapy (n=177)Unadjusted differenceAdjusted difference (95% CI)p valueMean (SD)nMean (SD)niHOT-33 6 months 46·6 (25) 161 45·6 (23) 154 1·0 −0·7 (−5·2 to 3·7) 0·743 12 months* 58·8 (27) 158 49·7 (25) 163 9·1 6·8 (1·7 to 12·0) 0·0093 EQ-5D-5L (utility) 6 months 0·544 (0·26) 144 0·573 (0·23) 147 −0·029 −0·042 (−0·088 to 0·005) 0·081 12 months 0·615 (0·25) 152 0·578 (0·24) 147 0·037 0·020 (−0·027 to 0·067) 0·397 EQ-5D VAS 6 months 67·8 (19·3) 145 70·3 (19·3) 145 −2·5 −2·1 (−5·7 to 1·4) 0·241 12 months 71·9 (20·7) 150 69·2 (19·4) 145 2·7 2·6 (−1·2 to 6·4) 0·180 SF-12 PCS 6 months 43·4 (7·0) 146 44·2 (6·6) 142 −0·8 −0·7 (−2·1 to 0·7) 0·304 12 months 45·1 (6·3) 145 44·2 (6·4) 132 1·0 1·1 (−0·2 to 2·5) 0·099 SF-12 MCS 6 months 42·1 (7·3) 146 42·1 (7·2) 142 −0·1 −0·1 (−1·5 to 1·3) 0·929 12 months 43·2 (7·1) 145 42·6 (6·9) 132 0·6 0·4 (−1·2 to 2·0) 0·589 iHOT-33=International Hip Outcome Tool. VAS=visual analogue score. PCS=physical component score. MCS=mental component score. *Primary outcome. In the prespecified subgroup analyses, the between-group difference for iHOT-33 was 5·0 (95% CI −1·2 to 11·3) in participants younger than 40 years and 10·9 (1·7 to 20·1) in those older than 40 years (pinteraction=0·3023) in favour of hip arthroscopy; the difference was 8·3 (95% CI 2·5 to 14·2) in patients with cam morphology, 1·1 (–11·5 to 13·7) in those with mixed cam and pincer morphology, and 4·0 (–14·6 to 22·7) in those with pincer morphology (pinteraction=0·5672), in favour of hip arthroscopy. Among patients who received their allocated intervention, there were 147 patient-reported adverse events (in 100 [73%] of 138 patients) in the hip arthroscopy group versus 102 (in 88 [60%]) of 146 patients in the personalised hip therapy group. The most frequently reported adverse event was muscle soreness, reported by 58 (42%) patients in the hip arthroscopy group and 69 (47%) patients in the personalised hip therapy group (table 3). At 12 months, seven serious adverse events had been reported by participating hospitals. Six of these were among the participants in the hip arthroscopy group: one patient was not discharged from the day surgery unit and required an overnight admission, one patient had scrotal haematoma requiring readmission, two patients had superficial wound infections that required oral antibiotics, one patient had a hip joint infection that required further surgery and ultimately a total hip replacement, and one patient had a fall that was unrelated to hip arthroscopy. One participant in the personalised hip therapy group developed biliary sepsis that was unrelated to treatment. There were no treatment-related deaths.Table 3Patient-reported adverse events Hip arthroscopy (n=138)*Personalised hip therapy (n=146)†p valueMuscle soreness at 6 weeks after intervention 58 (42%) 69 (47%) 0·40 Numbness in groin, leg, or foot 35 (25%) NA NA Hip pain or stiffness at 6 weeks after intervention 13 (9%) 8 (6%) 0·26 Unscheduled hospital appointments 13 (9%) 6 (4%) 0·096 Superficial wound problems 9‡ (7%) NA NA Hip joint infection 1 (1%) NA NA Fracture 0 NA NA Deep-vein thrombosis 0 NA NA Other adverse events potentially related to intervention 8 (6%; 2 numbness proximal thigh, 1 scrotal infection, 1 scrotal bruising, 1 labial swelling, 1 ankle pain, 1 erratic International Normalised Ratio, 1 nausea secondary to analgesia, 1 numbness to tip of tongue for 2 weeks after operation) 1 (1%; muscle spasms) 0·017 Other adverse events not related to intervention 18 (13%; 7 lower back pain, 2 knee pain, 2 road traffic collisions, 2 abdominal pain under investigation, 1 viral illness, 1 endometriosis, 1 chronic pain referred to rheumatologist, 1 skin discoloration, 1 multiple sclerosis) 0·17 NA=not applicable. *Six of 144 patients who received hip arthroscopy within 12 months did not return an adverse events form. †Eight of 154 patients who received personalised hip therapy only did not return an adverse events form. ‡Four patients required antibiotics. There was a low level of missing item-level data (eg, iHOT-33 0·6%) in all patient-reported outcome measures at all timepoints; after imputation for missing data, the adjusted estimate of treatment effect was similar, at 6·6 (95% CI 1·7 to 11·4) points in favour of hip arthroscopy. In a post-hoc analysis there was no significant difference in iHOT-33 at 12 months for patients in the hip arthroscopy group who were treated within 6 months of randomisation versus those treated 6 months or more after randomisation (0·9 [95% CI −10·7 to 8·8]). We assessed model assumptions, including assessment of quantile-quantile plots, which were deemed adequate. The mean cost of hip arthroscopy was £3042 (35% staff time, 28% surgical devices and anaesthetic drugs, 19% theatre-running costs, and 18% bed-day costs). Participants in the personalised hip therapy group attended a mean of six physiotherapy sessions (average duration of 30 min), generating mean total treatment costs of £155 per participant. The adjusted incremental cost of hip arthroscopy compared with personalised hip therapy during the 12-month follow-up was £2372, with incremental QALYs of −0·015 (representing a net QALY loss). Personalised hip therapy was more cost-effective than hip arthroscopy at 12 months (appendix). BODY.DISCUSSION: The UK FASHIoN study is the first randomised controlled trial to provide evidence that hip arthroscopy is effective in patients with femoroacetabular impingement syndrome. In this pragmatic trial, we found that iHOT-33 scores improved for patients in both groups; 12 months after randomisation, there was a mean adjusted difference of 6·8 points in the iHOT-33 score between patients allocated to receive hip arthroscopy and those allocated to receive personalised hip therapy, in favour of hip arthroscopy. This is a statistically significant difference that also exceeded the minimum clinically important difference for iHOT-33. These results are consistent with the hypothesis that hip arthroscopy is more clinically effective than best conservative care. There have been many observational studies showing benefit from hip arthroscopy; however, these studies generally did not have control groups for comparison, and are at high risk of bias.6 Results from a Cochrane systematic review done by members of our group11 showed that there had been no previous relevant randomised controlled trials. Since then, one recent randomised controlled trial26 has reported no difference between hip arthroscopy and conservative care. This study was small, was done in a military setting, with a single surgeon in a single centre, and with a very high rate of crossover (70%) from conservative care to hip arthroscopy.26 When the authors did a per-protocol comparison of those who had hip arthroscopy (n=66) with those who had conservative care (n=14), they reported that “power was lost making type II errors possible”.26 They concluded that “large cohorts across multiple sites are needed to make definitive conclusions”.26 Our trial is larger and therefore has greater power to detect between-group differences, and was done in 23 centres with a more generalisable patient population. There were no differences between groups in the secondary outcome measure of general health-related quality of life (EQ-5D-5L and SF-12). This finding could either be because treatment for femoroacetabular impingement syndrome does not have an effect on health-related quality of life or because the measures we used are not sufficiently sensitive to detect the changes that occur. A further possibility is that the trial was not sufficiently powered to detect changes in health-related quality of life. Adverse events in the hip arthroscopy group were more frequent than in the personalised hip therapy group. However, there was only one serious surgical complication in which a patient developed a hip joint infection. In one systematic review of 36 761 cases,27 hip arthroscopy had a reported complication rate of 3·3%, with a rate of major complications of 0·2%; our study findings are consistent with this. Our within-trial health economic evaluation suggests that hip arthroscopy is not cost-effective by comparison with personalised hip therapy. However, our economic models were only able to assess cost-effectiveness at 12 months from randomisation. This finding must also be set in the context of the high initial treatment costs of hip arthroscopy, the treatment timing (long delay in patients receiving hip arthroscopy, reducing the period of potential benefit during follow-up), and the period of economic inactivity during postoperative recovery (appendix). There could be long-term benefits from treatment that were not assessed in this economic analysis. We plan further follow-up points at 2, 3, 5, and 10 years, which will inform the lifetime cost-effectiveness of both surgery and personalised hip therapy, and whether treatment effects are maintained or if further treatments are required. Comparison of the rates of hip replacement in both groups will also help establish whether surgery affects the risk of osteoarthritis.1, 3 Strengths of this trial include the consent to participate rate among eligible patients (54%) and the follow-up rate (92%). Both of these values are high compared with similar trials in orthopaedics, and especially with trials of surgery versus no surgery, contributing to external and internal validity.28 The integrated qualitative research optimised recruitment, as it has done in other trials.15 This trial was thoroughly pragmatic, exploring the effectiveness of a strategy of offering hip arthroscopy compared with conservative care in the everyday reality of a national health service, where patients do not always receive or comply with the treatment they are offered, where surgeons and physiotherapists have varying levels of training, skill, and expertise, where postoperative care is variable, and where there are waiting lists for treatment. The large number of centres (n=23), surgeons (n=27), and physiotherapists (n=43) involved is a strength, which contributes to the generalisability of our findings. The comparator for this trial was personalised hip therapy, which we consider to be the best conservative care that can realistically be provided in the NHS for these patients. Personalised hip therapy was designed through international consensus and developed, supported, and tested in similar ways to other physiotherapist-led conservative care protocols.29 It meets the standards expected of a complex intervention in a randomised controlled trial, and was delivered by musculoskeletal physiotherapists who attended additional training and support events.6, 7, 30 Limitations of this trial include that participants and treating clinicians were not masked to treatment allocation. A blinded allocation trial, with a placebo control, would have been better suited to measuring the underlying effect of surgery. In our trial, the research question was whether hip arthroscopy or best conservative care was the most effective treatment strategy, leading to an inevitable absence of blinding. Data collection and analysis were done without revealing treatment allocation. An unexpected difficulty was the frequent delay in delivery of surgery for those patients allocated to hip arthroscopy. We had anticipated that this delay would be less than 3 months in most patients because when we designed the trial, there was a strongly enforced NHS target to treat patients within 18 weeks from referral to surgery. In fact, during the study, this target was a challenge in many hospitals. Patients allocated to hip arthroscopy therefore often had longer times to treatment, and because the primary outcome was measured 12 months after randomisation, these patients were often still within a few months (and in some cases a few weeks) of their operation when we measured the primary outcome. Because of these delays, patients in the hip arthroscopy group had, on average, less time to recover before the primary outcome measurement than did patients in the personalised hip therapy group. We compared the outcome of hip arthroscopy participants who had surgery in the first 6 months after randomisation with those who had surgery in the second 6 months. There was no significant difference between these groups, suggesting that the systematic difference in time to treatment between groups does not account for the treatment effect. Our inferences about the effectiveness of hip arthroscopy compared with personalised hip therapy are limited to data collected 12 months after randomisation; long-term follow-up is required to establish if this effect is maintained and if further treatments are required. The fact that not all surgery or personalised hip therapy was deemed to be of a high fidelity is also a reflection of the real-world setting in which this trial was done. Some surgery was not satisfactory, and some participants allocated to personalised hip therapy did not engage with it or complete it: our fidelity assessment showed high-fidelity treatment in 87% of patients receiving hip arthroscopy and 70% of patients receiving personalised hip therapy. However, these proportions are comparable with other studies and reflect the pragmatism of our trial.31 We took great effort to minimise crossover in our trial, using techniques developed in our feasibility study. No participants allocated to receive hip arthroscopy received personalised hip therapy; 14 participants allocated to personalised hip therapy subsequently changed their mind and decided to have surgery within 12 months. We do not believe that these crossovers can account for the results of this trial; indeed, we would expect such crossovers to dilute and so reduce our estimate of the real underlying effect of hip arthroscopy. Personalised hip therapy is believed to work by improving muscle control, strength around the hip, and movement patterns, leading to the avoidance of hip impingement. Surgery is thought to work by reshaping the bone to prevent impingement and by treating painful injuries to articular cartilage and labrum. In our trial, the observed effect of hip arthroscopy over conservative care might be attributable to the surgical procedure, the placebo effect of surgery (given the unblinded nature of this trial), post-surgical rehabilitation, or a combination of these factors. The results of our subgroup analysis of patients with only cam morphology are suggestive of an increased treatment effect of hip arthroscopy compared with other shapes. This finding would support the idea that the removal of a cam shape has a specific therapeutic effect. The low number of patients with pincer or mixed cam and pincer morphology in this study means we are less certain about the influence of reshaping the acetabular rim. Ultimately, we do not fully understand the mechanism of benefit from hip arthroscopy or personalised hip therapy. Future research should focus on investigating these mechanisms further, and which patients would benefit most from hip arthroscopy or personalised hip therapy. We have shown that offering hip arthroscopy to patients with femoroacetabular impingement syndrome led to better patient-assessed function 12 months after randomisation, compared with best conservative care. This improvement comes at a cost; our study does not show cost-effectiveness of hip arthroscopy compared with conservative care within the first 12 months.

TITLE: Behaviour of the Foramen Ovale Flow in Fetuses with Intrauterine Growth Restriction ABSTRACT.BACKGROUND: Foramen ovale (FO) flow may be altered in IUGR. This study was designed to test this hypothesis. ABSTRACT.METHODS: Forty pregnant women (24–38 weeks) were divided into 3 groups: group I (IUGR), group II (adequate growth and maternal hypertension), and group III (normal controls). Impedance across the FO was assessed by the FO pulsatility index (FOPI): (systolic velocity − presystolic velocity)/mean velocity. Statistical analysis utilized ANOVA, Tukey test, and ROC curves. ABSTRACT.RESULTS: Mean FOPI in IUGR fetuses (n = 15) was 3.70 ± 0.99 (3.15–4.26); in the group II (n = 12), it was 2.84 ± 0.69 (2.40–3.28), and in the group III (n = 13), it was 2.77 ± 0.44 (2.50–3.04) (p=0.004). FOPI and UtA RI were correlated (r = 0.375, p=0.017), as well as FOPI and UA RI (r = 0.356, p=0.024) and, inversely, FOPI and MCA RI (r = −0.359, p=0.023). ABSTRACT.CONCLUSIONS: The FO flow pulsatility index is increased in fetuses with IUGR, probably as a result of impaired left ventricular diastolic function. BODY.1. INTRODUCTION: Intrauterine growth restriction (IUGR) is a significant clinical problem, affecting up to 10% of all pregnancies [1] and even 15% of all monochorionic twin pregnancies [2], with high perinatal morbidity and mortality rates due to fetal hypoxia [3, 4]. Placental insufficiency is the etiology in most cases [5, 6], although infections, congenital anomalies, and drug misuse are other associated conditions [7]. Monitoring the consequences of fetal hypoxia is the basic obstetric management, given that the only current treatment for IUGR is delivery [7, 8]. The fetal heart is a central organ in adaptive mechanisms to hypoxia, and cardiac dysfunction is recognized as the pathophysiologic determinant of clinical deterioration in both early- and late-onset IUGR [4]. Biophysical profile and ductus venosus impedance become abnormal only in advanced stages of fetal compromise [7], and for this reason, alternative parameters, in particular cardiac function, could provide earlier markers with higher sensitivity [7, 9]. In normal pregnancy, a progressive decrease in umbilical artery (UA) impedance occurs, which allows appropriate fetal cardiovascular development [4]. IUGR fetuses have abnormal placental changes, with increased placental vascular resistance and progressive deterioration of the UA flow [10]. UA Doppler is indicated for early detection of placental insufficiency [11]. Fetal circulatory response primarily benefits the systemic cardiac output (CO), providing an adequate oxygen supply to vital organs [12, 13]. With progression of fetal pulmonary and systemic vasoconstriction, an increased right ventricular afterload and a shift of cardiac output to the left ventricle occur [8], with impact on left diastolic function and possible increased impedance to flow through the foramen ovale (FO) [14]. Due to its triphasic flow pattern, the vascular pulsatility index (PI) may represent its impedance, as already demonstrated in other situations such as gestational diabetes [15]. The analysis of the foramen ovale flow dynamics in fetuses with IUGR has not been previously assessed. The purpose of this study was to compare the foramen ovale pulsatility index (FOPI) in IUGR fetuses with the FOPI in fetuses classified as of appropriate growth, with and without maternal hypertension. Correlations of the FOPI with maternal, fetal, and placental Doppler indices were tested. BODY.2. METHODS: A cross-sectional, controlled, nonblinded study of 40 single fetuses with 24 weeks of gestational age (GA) or more was designed. The sample included 15 growth-restricted fetuses (group I), 12 fetuses with normal weight for gestational age from hypertensive mothers (group II), and 13 fetuses with normal weight for gestational age from healthy mothers (group III). Maternal hypertension was classified according to Guidelines of the American College of Obstetricians and Gynecologists [16]. Fetuses with other abnormalities or fetuses whose mothers used drugs or tobacco were excluded from the study. All pregnant women provided written informed consent to participate in the study, which was approved by the Medical Research Ethics Committee of the Institute of Cardiology of Rio Grande do Sul. Gestational age was determined in all fetuses by first trimester ultrasound. After 24 weeks, a morphological ultrasound was performed for weight estimation (according to the Hadlock method) [17], placental assessment, amniotic fluid volume determination, and flow velocimetry of the umbilical, middle cerebral, and uterine arteries. Under fetal apnea, the resistance index (RI) was obtained by Doppler flow analysis and determined by the ratio (systolic velocity − diastolic velocity)/systolic velocity. The amniotic fluid index was measured by the sum of the pockets of the four quadrants of the maternal abdomen. IUGR with placental insufficiency was defined by fetal weight below the 10th percentile for gestational age in combination with abnormal Doppler indices (either umbilical artery RI > 95th centile, middle cerebral artery RI < 5th centile, or uterine artery RI > 95th centile for gestational age). Fetal echocardiography was performed using an Acuson Aspen (Acuson, Mountain View, CA, USA) ultrasound system, with a multifrequencial transducer (3 to 5 MHz). Cardiac structural assessment used segmental sequential analysis. Flow in the FO was obtained at a four-chamber view, with the pulsed Doppler sample volume placed at the left atrial surface of the orifice in the central portion of the color flow mapping, with an angle of less than 20° [15, 18]. Flow impedance was assessed by the pulsatility index, obtained by the ratio (systolic velocity − presystolic velocity)/mean velocity [19] (Figure 1). The statistical package SPSS version 15.0 (SPSS Inc., Chicago, IL) was used for data analyses. Quantitative analyses were reported as mean ± standard deviation (SD). Analysis of variance (ANOVA) was used for each variable. The Tukey test was applied for the individual assessment of each group when significant differences were present. The chi-square test of the observed frequencies was used for qualitative analyses. The correlation of the FOPI with fetal arteries' RI was tested by the Pearson test. The critical significance level was p < 0.05. The sample size was not calculated since no previous studies were available to assess differences. Nevertheless, the study power was calculated based on the pulsatility index of the foramen ovale, considering a significance level of 5%, standard deviations of 0.44 and 0.99, and a difference of 0.93 in the pulsatility indices of groups I and III, achieving a study power of 84%. The reproducibility of measurements of the FOPI was tested. Intraobserver variability was assessed in twelve nonconsecutive normal fetuses by repeating the measurements on two occasions (2 days apart) with the same conditions. Interobserver variability was also assessed, with measurements repeated in 10 normal fetuses on the same day by a second observer blinded to the results of the first examination. Intraclass correlation coefficients were calculated to measure the degree of consistency among measurements. The Bland–Altman plots were created to show the mean of differences between measurements. BODY.3. RESULTS: Table 1 presents the characteristics of the sample, showing that the groups were comparable except for maternal age and body mass index (BMI) in group II when compared to the other two groups. Gestational age ranged from 24 to 38 weeks. Minimum maternal age was 14 years and maximum 38 years. Doppler features of study groups are shown in Table 2. The FOPI (mean ± standard deviation) in group I was 3.70 ± 0.99 (95% confidence interval: 3.15 to 4.26), 2.84 ± 0.69 (95% CI: 2.40 to 3.28) in group II, and 2.77 ± 0.44 (95% CI: 2.50 to 3.04) in group III (p=0.004) (Figure 2). Pearson analysis of the FOPI showed a positive correlation of the FOPI with the UA (r = 0.356, p=0.024) and the UtA (r = 0.375, p=0.017). The correlation was negative with the MCA (r = −0.359, p=0.023) (Figure 3). To assess the agreement between two sets of measurements, intraclass correlation coefficients for FOPI measurements were estimated: intraobserver variation was 0.8035 (95% CI: 0.5002 to 0.9312) and interobserver variation was 0.8227 (95% CI: 0.4371 to 0.9528). The Bland–Altman plots were created (Figure 4). BODY.4. DISCUSSION: In this study, we assessed the foramen ovale flow dynamics in fetuses with IUGR and with adequate growth of both normotensive and hypertensive women. It was observed that IUGR fetuses had an increased impedance to flow through the FO, represented by an increased FOPI compared to control groups, probably as a result of impaired diastolic function. The same effect has been demonstrated in fetuses of diabetic mothers (FDMs) with myocardial hypertrophy, where the FOPI was higher than that in normal fetuses [15]. Several studies have demonstrated changes in diastolic function in FDMs, being the ductus venosus PI [20] and the pulmonary vein PI [21] significantly higher in fetuses with myocardial hypertrophy, probably due to a lower ventricular compliance. The mobility of the septum primum is lower in fetuses with IUGR [22] and myocardial hypertrophy [23] when compared to normal fetuses, due to a decreased ventricular compliance. The shortening fraction of the left atrium was also shown to be decreased in fetuses with myocardial hypertrophy, presenting a negative correlation with the septal thickness [24]. Left ventricular myocardial diastolic velocities by tissue Doppler imaging (TDI) were shown to be significantly higher in FDM, irrespective of the presence of cardiac hypertrophy, suggesting that maternal diabetes is associated with changes in left ventricle diastolic function even without myocardial hypertrophy [25]. TDI evaluation of IUGR fetuses showed that myocardial early and late diastolic velocity ratios were higher in IUGR in lateral and septal mitral annulus, when compared with normal growing fetuses [9]. A subsequent study [26] confirmed these findings. Both studies concluded that TDI can probably be a more sensitive method for detection of diastolic cardiac dysfunction in IUGR fetuses than conventional mitral and tricuspid valve Doppler [9, 26]. In a recent study in rabbits, cardiac weight reduction was demonstrated in response to IUGR, due to a decrease in the number of cardiomyocytes in both ventricles. However, an increase in the mean volume of cardiomyocytes occurred in the left ventricle. This demonstrates that the right and left ventricles respond differently to placental insufficiency [27]. IUGR fetuses have impaired ventricular filling, with higher atrioventricular valve E/A ratios, lower aortic and pulmonary artery systolic peak velocities, left ventricular CO increase, and right ventricular CO decrease. These hemodynamic changes favor flow into the left ventricle in order to maintain cerebral perfusion [7, 18]. Therefore, in the early stages of the disease, adequate levels of oxygen and substrates are maintained, despite the reduction in placental transfer. During the process of centralization, fetal changes occur in cardiac afterload, decreasing left ventricle afterload due to cerebral vasodilatation and increased systemic vasoconstriction [28]. Furthermore, hypoxia may impair myocardial contractility, while polycythemia may increase blood viscosity [7]. In fetuses with IUGR, a faster presystolic left-to-right flow velocity through the FO is in accordance with a study that assessed flow in fetuses with left ventricle hypoplasia or left side obstruction, in which there was increased left atrial pressure [29]. Umbilical and uterine artery flow RIs were not different between groups. Correlation analysis of the RI of maternal and fetal flows with the FOPI showed a positive weak correlation with the UA and the UtA and a negative weak correlation with the MCA. Nonstratification according to the severity of placental insufficiency in group I could be a limitation and may explain these data, as a stronger correlation would be expected with more severe fetal compromise. Left diastolic function assessed by the FOPI could already be altered in cases of IUGR with placental insufficiency in early stages. Turan et al. [28] assessed the time of onset of placental dysfunction in fetuses with IUGR, and in two of the three study groups, patients were recruited when there was no placental dysfunction. This situation was diagnosed only two to four weeks later. The fetal cardiac output is redistributed during hypoxia, promoting flow to vital organs like brain, heart, and adrenal glands [30–32] and justifying the MCA resistance decrease associated with cardiac diastolic dysfunction assessed by FOPI. The possibility of maternal hypertension without IUGR is supported by other authors. Grisaru-Granovsky et al. [33] showed that the presence of IUGR in fetuses of hypertensive mothers was not correlated with worsening of the hypertensive disorder but reflected the individual predisposition of fetuses to abnormal development. Our study had a small sample size of group I, not allowing categorization in relation to the presence of associated maternal hypertensive disorder. This study has limitations. It was not blind, allowing the potential occurrence of a mensuration bias. Being a cross-sectional study, it was not possible to assess the sequential changes in flow through the FO with the evolution of pregnancy. The lack of stratification according to the severity of fetal compromise and maternal hypertensive disorder in group I has already been mentioned. It was observed that group II had higher maternal ages and BMI than the other groups, but these are not expected to primarily interfere with fetal cardiac hemodynamics. Despite the occurrence of oligohydramnios in one-third of the IUGR sample, technical aspects of assessment of the FOPI did not represent any limitation, as demonstrated by the strength of agreement between two sets of measurements obtained by trained operators. It has been reported that IUGR fetuses may show different outcome patterns [28]. For this reason, it is important to perform serial tests in an effort to promote birth with the lowest possible morbidity and mortality. The use of other techniques, such as fetal echocardiography, may help monitoring IUGR fetuses [34]. The present study suggests that the presence of an increased FOPI, especially above 2.95, may suggest abnormalities in fetal diastolic heart function, thus potentially influencing the overall obstetric management.

TITLE: Preemptive use of etodolac on tooth sensitivity after in-office bleaching: a randomized clinical trial ABSTRACT.ABSTRACT: ABSTRACT.PURPOSE: This study determined the effectiveness of the preemptive administration of etodolac on risk and intensity of tooth sensitivity and the bleaching effect caused by in-office bleaching using 35% hydrogen peroxide. ABSTRACT.MATERIAL AND METHODS: Fifty patients were selected for this tripleblind, randomized, crossover, and placebo-controlled clinical trial. Etodolac (400 mg) or placebo was administrated in a single-dose 1 hour prior to the bleaching procedure. The whitening treatment with 35% hydrogen peroxide was carried out in two sessions with a 7-day interval. Tooth sensitivity was assessed before, during, and 24 hours after the procedure using the analog visual scale and the verbal rating scale. Color alteration was assessed by a bleach guide scale, 7 days after each session. Relative risk of sensitivity was calculated and adjusted by session, while overall risk was compared by the McNemar's test. Data on the sensitivity level of both scales and color shade were subjected to Friedman, Wilcoxon, and Mann-Whitney tests, respectively (α=0.05). ABSTRACT.RESULTS: The preemptive administration of etodolac did not affect the risk of tooth sensitivity and the level of sensitivity reported, regardless of the time of evaluation and scale used. The sequence of treatment allocation did not affect bleaching effectiveness, while the second session resulted in additional color modification. The preemptive administration of etodolac in a single dose 1 hour prior to in-office tooth bleaching did not alter tooth color, and the risk and intensity of tooth sensitivity reported by patients. ABSTRACT.CONCLUSION: A single-dose preemptive administration of 400 mg of etodolac did not affect either risk of tooth sensitivity or level of sensitivity reported by patients, during or after the in-office tooth bleaching procedure. BODY.INTRODUCTION: Tooth whitening is a simple and non-invasive treatment commonly carried out to reestablish smile aesthetics. High success rates have been demonstrated for bleaching techniques applying 35% hydrogen peroxide (HP35%) 2 . Hydrogen peroxide (H2O2)-based bleaching agents at high concentrations (typically 15-38%) are currently used for in-office techniques due to their high oxidizing ability 12 , 25 . However, the low molecular weight of H2O2 allows its penetration across the entire dentin tissue, reaching the pulp chamber and promoting damage of pulp stem cells, which is reported by patients as tooth sensitivity 15 , 29 . Prior clinical trials have reported absolute risk of tooth sensitivity as high as 95% when highly concentrated H2O2 is used for in-office tooth bleaching 9 , 20 , 23 , 25 . Thus, the preemptive use of desensitizer agents 3 , 30 or anti- inflammatories 7 , 22 , 23 , 27 has been proposed to reduce the risk of post-bleaching tooth sensitivity. Only the former significantly decreased tooth sensitivity; however, the application of desensitizers, when not incorporated into the bleaching gel, adds an extra step to the bleaching protocol, which is contrary to a clinician's need for simplification. On the other hand, the preemptive use of anti-inflammatories does not increase the number of steps in the bleaching protocol. Unfortunately, prior studies evaluating etoricoxib, ibuprofen, or dexamethasone were unable to demonstrate any beneficial effect on tooth sensitivity caused by tooth bleaching 7 , 13 , 22 , 23 , 28 . Etoricoxib and ibuprofen are grouped as class II drugs by the Biopharmaceutical Classification System (BCS), presenting low solubility and high permeability, which can hinder their absorption and create bioavailability mismatch during the bleaching procedure 28 . Moreover, anti-inflammatory drugs may have a specific action over inflammatory mediators (bradykinin) and the neurotransmitter (substance P) of tooth pain caused by dental bleaching 6 . Among non-steroid anti-inflammatory drugs (NSAIDs), etodolac demonstrated efficacy on the control of prostaglandins and bradykinins 18 . Nevertheless, there is no clinical evidence regarding the use of etodolac on the reduction of tooth sensitivity caused by in-office bleaching. Thus, this study aimed to evaluate the effectiveness of the preemptive prescription of etodolac on risk of tooth sensitivity during and after in-office bleaching treatment. The first hypothesis evaluated was that etodolac would reduce both level and risk of tooth sensitivity when administrated in a single dose prior to in-office bleaching. The second hypothesis tested was whether the use of etodolac would reduce tooth sensitivity with no effect on tooth bleaching. BODY.MATERIAL AND METHODS: This clinical trial followed the CONSORT statements and was approved by the Scientific Review Committee and by the Committee for the Protection of Human Subjects of the local university (CAAE 37578714.4.0000.5546), and registered at clinicaltrials.gov under the number NCT02881619. BODY.TRIAL DESIGN: This study was a randomized, triple-blind, placebo- controlled clinical trial with a crossover design. Patients included signed an informed consent form and were submitted to two in-office bleaching sessions receiving placebo (control) or etodolac prior to the bleaching procedure, while different treatments were allocated for each session. A one-week interval ("washout") in-between sessions was established. The study was conducted at the clinic of the School of Dentistry of the local university from November 2014 to July 2015. BODY.PARTICIPANTS: Patients from 18 to 35 years old with good oral health were included in this clinical trial. From the patients who received placebo/etodolac, 6 were men and 19 were women, and for etodolac/placebo, 12 were men and 13 were women; the average age was 23 years; 64% were women. Patients with any of the six upper anterior teeth with caries, restoration, severe discoloration (e.g., stains caused by tetracycline), enamel hypoplasia, gingival recession, dentin exposure, pulpitis, or endodontics were excluded, as well as smokers. Participants submitted to previous bleaching procedures, with prior tooth sensitivity, known allergy to any component of the medication used in the study, and pregnant or breastfeeding women were also excluded. An air stream was applied to teeth to verify the presence or absence of sensitivity (none or zero). Only patients presenting all six upper anterior teeth with shade mismatch of 2.5 M2 (Vita Bleach guide 3D-Master scale, Vita-Zahnfabrik, Bad Sackingen, Germany) - A3.5 Vita Classical equivalence - or darker were included. BODY.SAMPLE SIZE CALCULATION: The sample calculation was based on the primary binary outcome (sensitivity risk 24 hours after the procedure) for superiority trial. The power of the test was set at 80%, considering a type I error of 0.05 and risk of tooth sensitivity of 90%, based on a prior study using a similar bleaching agent 23 ; moreover, a reduction around 30% was expected after treatment. The calculation resulted in fifty patients. BODY.RANDOMIZATION: A randomized list was computer-generated by a person not involved in intervention or evaluation. Participants were defined as blocks in the randomization process, in which the sequence of treatment (placebo or etodolac) was randomly set for each block by computer-generated tables (www.sealedenvelope.com). The sequence was inserted into sealed envelopes numbered from 1 to 50 that were opened by the operator only at the moment of intervention. Patients were numbered according to the sequence of enrollment. Neither the participant nor the operator knew the group allocation, determining a blinded protocol. BODY.BASELINE EVALUATION: Prior to the bleaching procedure, teeth were cleaned using rubber cups associated with pumice and water. The shades of upper incisors and canine teeth (13, 12, 11, 21, 22, and 23) were assessed on a baseline using the bleach guide scale. Color was analyzed by two previously calibrated evaluators. Both evaluators presented superior color matching competency according to the ISO (International Organization for Standardization)/TR 28642:2011. Shade tabs selected were converted to scores ranging from 1 (whiter shade – 0M1) to 15 (darker shade – 5M3). Considering a possible effect of dental anxiety on the sensitivity reported by patients, the Corah's Dental Anxiety Scale was used to determine the level of anxiety of each patient related to the procedure 17 . Each answer to the survey instrument was scored on a scale from 1 to 5 (four questions) and the sum of scores was used to determine the level of anxiety: low was under 12, moderate was between 12 and 14, and high was over 14. BODY.INTERVENTION: One hour before each bleaching session and right after the prophylaxis, patients received a capsule containing 400 mg of NAISE etodolac (Flancox™, Apsen Farmaceutica S/A) or 400 mg of placebo (inert content) according to randomization. Capsules had the same appearance and were manufactured by a person not involved in intervention or evaluation. They were placed into two bottles identified by letters according to the treatment. Neither the operators responsible for intervention and evaluation nor the patients knew the content of each capsule. The color evaluation was verified, and the light- cured resin dam was applied (Top Dam, FGM, Joinville, SC, Brazil) and polymerized (Radii-cal, SDI, Bayswater, Australia) on the gingival tissue corresponding to the teeth to be bleached. A 35% hydrogen peroxide-based bleaching agent (Whiteness HP Blue, FGM, Joinville, SC, Brazil) was mixed and applied to the buccal surface of teeth for 40 minutes. After that, the bleaching agent was removed. A second session was carried out after a week following the same procedures. At this time, the patient received a single-dose capsule containing etodolac or placebo (different from the ones received at the first session), one hour before the procedure. During the bleaching treatment, patients were advised not to ingest colored food and beverages. BODY.EVALUATIONS: Tooth sensitivity reported by patients was assessed using a visual analog scale (VAS) and a verbal rating scale (VRS). The VAS consisted of a 10-cm long scale ranging from green (absence of pain) to red (unbearable pain). Patients set their level of sensitivity by pointing to the color corresponding to the pain level, while the distance from this point to the green border was recorded. For the VRS, patients reported their level of sensitivity based on scores: 0= none; 1 = mild; 2= moderate; 3= considerable; and 4= severe. Tooth sensitivity was assessed during bleaching, immediately after removing the bleaching agent, and after 24 hours. For this last assessment, only the VRS was used due to the difficulty of patients to fill the VAS at home. One week after each session, tooth color was evaluated again using the same procedure described previously. BODY.STATISTICAL ANALYSIS: Demographic data from patients were analyzed to determine age, gender, and anxiety level for each allocation sequence. Comparisons among allocation sequences were performed by Mann-Whitney (age), Fisher's exact (gender), and chi-square (anxiety level) tests. Based on the presence of any tooth sensitivity (VRS scores different from 0), the absolute risk, odds ratio, and relative risk were calculated regarding the treatments for each moment of bleaching evaluation/ session, as well as the confidence intervals (95%). For each moment, differences on presence/absence ratios were analyzed by Fisher's exact test. For the overall risk related to each treatment, odds ratio was adjusted to the independent variable of "session of bleaching" using Mantel-Haenszel statistics. The homogeneity of odds ratios was analyzed by Breslow-Day and Tarone's tests. Next, the estimated odds ratio was converted to relative risk and the overall presence/absence ratios were analyzed by the McNemar's test, considering the study design (crossover). For the VRS, data from scores observed at each moment of bleaching evaluation/session were submitted to the Mann-Whitney rank sum test. Despite the measurement of tooth sensitivity using the VAS, which provided a continuous outcome, data assessed with this scale did not show normal distribution (Shapiro-Wilk's test). Thus, data from the VAS were also analyzed by the Mann-Whitney rank sum test, performing one test per time of evaluation. For color evaluation, comparisons among sequences of treatment were performed using the Mann-Whitney rank sum test. Friedman test followed by Dunn's post hoc test were used to analyze the difference between the moments of evaluation for each sequence of treatment. All statistical analyses were performed adjusting the initial significance level (α=0.05) by the Bonferroni correction. BODY.RESULTS: Figure 1 shows the flow chart of patients assessed for eligibility, who were included in the study and analyzed. Table 1 shows the demographic characteristics of patients allocated for each sequence of treatment. Regarding anxiety, 88% of patients had a low level and only 2% had a high level (p=0.236). There was no difference among the sequences of treatment for any demographic characteristic analyzed (age: p=0.089 and gender: p=0.140). Figure 1BODY.FLOW CHART OF PATIENTS: Table 1BODY.PROFILE OF PATIENTS INCLUDED IN THE STUDY ALLOCATED TO EACH SEQUENCE OF TREATMENT: Age (years)Median (1st / 3rd quartiles)p-value Total 23.0 (21.0/26.0) Placebo/Edotolac 23.8 (21.0/25.5) p * = 0.089 Edotolac/Placebo 25.0 (22.0/28.0) Gender Total n (%) Male 18 (36.0%) Female 32 (64.0%) Placebo/Edotolac p ** = 0.140 Male 6 (24.0%) Female 19 (76.0%) Edotolac/Placebo Male 12 (52.0%) Female 13 (48.0 %) Level of anxiety Total n (%) Low anxiety 44 (88.0%) Moderate anxiety 5 (10.0%) High anxiety 1 (2.0%) Placebo/Edotolac p *** = 0.236 Low anxiety 21 (84.0%) Moderate anxiety 4 (16.0%) High anxiety 0 (0.0%) Edotolac/Placebo Low anxiety 23 (92.0%) Moderate anxiety 1 (4.0%) High anxiety 1 (4.0%) *Mann-Whitney rank sum test; **Fisher Exact test; ***Chi-square test. Table 2 shows the results of tooth sensitivity risk. The treatment that patients received prior to the bleaching procedure did not affect the risk to sensitivity at any of the moments of evaluation (during and immediately after: p=1.0; after 24 hours: p=0.683). Figure 2 shows the results for level of sensitivity assessed by the VRS. Treatment did not affect level of sensitivity, regardless of the moment of evaluation. Similar results were observed when the VAS was used (Figure 3). Table 2BODY.RESULTS OF RISK OF TOOTH SENSITIVITY OBSERVED FOR EACH TREATMENT: SessionMoment of evaluationDuringImmediately after24 h after Treatment Edotolac Placebo Edotolac Placebo Edotolac Placebo 1st session Presence of sensitivity (yes/no) (10/15) (11/14) (9/16) (10/15) (3/22) (2/23) Odds ratio (95% CI) 0.52 (0.17 – 1.61) 0.84 (0.27 – 2.65) 1.57 (0.24 – 10.30) Relative risk (95% CI) 0.71 (0.40 – 1.29) 0.90 (0.44 – 1.83) 1.50 (0.27 – 8.22) p-value* 0.396 1.000 1.000 2nd session Presence of sensitivity (yes/no) (8/17) (5/20) (6/19) (4/21) (1/24) (1/24) Odds ratio (95% CI) 1.88 (0.52 – 6.85) 1.66 (0.41 – 6.79) 1.00 (0.59 – 16.93) Relative risk (95% CI) 1.60 (0.61 – 4.22) 1.50 (0.48 – 4.68) 1.00 (0.06 – 15.12) p-value* 0.520 0.725 1.000 Average Odds ratio (95% CI)** 0.92 (0.40 – 2.09) 1.11 (0.46 – 2.67) 1.37 (0.29 – 6.51) Relative risk*** 0.95 1.08 1.34 p-value**** 1.000 1.000 0.683 *Fisher exact test; **Mantel-Haenszel common odds ratio estimate; ***Based on odds ratio estimated; ****McNemar's test. The cut-off value of type I error (α=0.0056) was adjusted by Bonferroni correction. Figure 2BODY.SCORES OF TOOTH SENSITIVITY FROM THE VRS (0 TO 4): Figure 3BODY.LEVEL OF TOOTH SENSITIVITY ASSESSED WITH VAS (CM): In regards to bleaching effectiveness, the bleaching procedure carried out in this study was able to significantly reduce the shade scores from the bleach guide, while the second session resulted in additional bleaching effect (Figure 4). The sequence of treatment did not affect bleaching effectiveness. Figure 4BODY.SCORES FROM THE BLEACH GUIDE SCALE: BODY.DISCUSSION: Tooth bleaching performed by patients at home using low-concentration peroxides has been reported as the first-choice technique for vital bleaching, and probably the most widely used one 5 , 11 . However, procedures using high-concentration bleaching agents applied in office by clinicians remain an important protocol used to bleach discolored teeth for specific indications of tooth bleaching. This protocol is separate and intended to aid patients who cannot adapt to the use of home-based bleaching trays and who have contraindications related to the gastric system, because it reduces the risk of gel intake 14 , and to control the risk factor for developing gingival irritation 10 . In-office bleaching is also indicated to patients requiring faster results, while this technique can be combined with at-home bleaching 26 . In-office techniques have demonstrated high bleaching effectiveness using high-concentration hydrogen peroxides 26 . However, high-concentrated bleaching agents also result in increased tooth sensitivity reported by patients during and up to 24 hours after the bleaching procedure, which is the main adverse effect related to in-office tooth bleaching 9 , 20 , 21 . Even though tooth sensitivity is related to the inflammatory process of pulp tissue 20 , the findings of this study showed that the preemptive use of etodolac in a single dose did not affect the risk and level of tooth sensitivity caused by in-office bleaching. As expected, preemptive administration of etodolac also did not affect the bleaching results. Thus, the first hypothesis of the study was rejected and the second one was accepted. Unlike the tooth sensitivity typically reported by patients presenting teeth with dentin exposure, which relates mainly to thermal stimuli, bleached teeth can hurt even in the absence of any stimulus, showing that the pain mechanism related to peroxides is different from other types of tooth pain 20 . Moreover, the sensitivity caused by tooth bleaching tends to increase within a few hours following the bleaching procedure, when most patients described the pain as a "twinge" or "shock-like." It has been demonstrated that the oxidizing agents used during the bleaching procedure cause a reduction on metabolism, viability, and cell proliferation 20 , allowing to increase the expression of inflammatory mediators, such as substance P and bradykinin, which is a vasoactive peptide released by nerves resulting in a neurogenic inflammation 6 . Thus, a preemptive administration of anti-inflammatories could be a reasonable approach to reduce tooth sensitivity associated with bleaching procedures. However, in this study, sensitivity was not lower during and 24 hours after the bleaching procedure. Unfortunately, the peak of tooth sensitivity was not measured in this study, even though this outcome could allow to assess a possible effect of etodolac on sensitivity following the end of the bleaching procedure. A prior study demonstrated that etodolac presents higher effectiveness on bradykinin inhibition than other drugs commonly used to control the inflammatory process, motivating its use in this study 18 . Regarding pharmacokinetics, etodolac reaches its maximal plasma concentration around 1 to 2 hours after its administration 4 . Thus, it was expected that maximal plasma concentration would be reached during the bleaching procedure. Unfortunately, no effect of the preemptive administration of etodolac on the reported tooth sensitivity was observed at this time of evaluation for both sessions. Despite its demonstrated efficacy on inflammation control, the analgesic effect of etodolac increases when its administration is repeated, whereas a single dose prior to the bleaching procedure seems insufficient to prevent tooth sensitivity 19 . Another important observation regarding pharmacokinetics is that a crossover design was used in this study with a 1-week interval period. Considering that the half-life of etodolac after oral administration is around 13 hours 16 , it is expected to find no residual effect after 1-week. Moreover, the crossover design avoids bias related to pain thresholds of patients 1 . Participants included in this study were predominantly young females presenting low level of anxiety. All these demographics characteristics of the studied population might be associated with differences in pain thresholds. Higher tooth sensory threshold has been demonstrated in males due to differences in crown diameters of teeth and underlying mechanisms such as neurological differences or behavior aspects 8 . Regarding the age of participants, a recent review did not find any relation between age and the risk to or level of tooth sensitivity 27 . However, it is important to emphasize that most participants from trials included in that review were under 30 years old 27 . Another important demographic aspect assessed in this study was the participants' level of dental anxiety prior to bleaching procedures. It has been demonstrated that dental anxiety is a strong predictor of pain and that anxious participants are prone to develop painful responses 24 . In this research, almost 90% of participants presented low anxiety prior to bleaching procedures, which can be justified by the low invasive aspect of intervention, despite the patients' concern about tooth sensitivity. In fact, despite 66% of participants reporting various level of tooth sensitivity (high risk), the actual level of sensitivity reported was low (medians below moderate at VRS, and means lower than 2 at VAS). In addition to the evaluation of tooth sensitivity, we also assessed color alteration promoted by bleaching procedures. The data analysis of color evaluation used the sequence of allocation instead of the treatment (placebo or etodolac). If we had used treatment, different participants between the first and second sessions would be compared for the same treatment, which would have impaired correct color changes assessment. Moreover, the main aim of color evaluation was to show the effectiveness of the bleaching technique used. In our study, the protocol that was carried out resulted in significant bleaching effect, while an additional color alteration was achieved at the second session. In addition, the last color evaluation was performed a week after the last bleaching session of this study, while longer times may be required for color stabilization 21 . However, a shorter time was used because the tooth sensitivity reported by patients was the main outcome of this trial. It has been demonstrated that two sessions of in-office tooth bleaching results in a mean change of 5.3 (± 2.8) units on shade guides 26 , which is similar to the average color change achieved in this research. Factors such as patient's age and color at baseline have been strongly associated with bleaching effectiveness, while young patients and darker teeth show more pronounced color changes 27 . We found no difference in baseline regarding these parameters between the sequence of allocation, while the inclusion of young participants presenting all teeth darker than 2.5 M2 (score 7) favored obtaining significant color bleaching. In conclusion, the preemptive administration of etodolac in a single dose 1 hour prior to the bleaching procedure was unable to reduce both risk and level of sensitivity caused by in-office bleaching. A limitation of this study was that the preemptive treatment was administrated only for a young population (average age of 23 years), with prevalence of female patients, while different results can be observed for other demographic profiles 27 . BODY.CONCLUSIONS: The preemptive administration of a single dose of etodolac previously to the two bleaching sessions with 35% hydrogen peroxide did not affect tooth color change, risk of sensitivity and level of pain reported by the patients (during the sessions, immediately after, and 24 h after sessions).

TITLE: Optimal Mode of clearance in critically ill patients with Acute Kidney Injury (OMAKI) - a pilot randomized controlled trial of hemofiltration versus hemodialysis: a Canadian Critical Care Trials Group project ABSTRACT.INTRODUCTION: Among critically ill patients with acute kidney injury (AKI) needing continuous renal replacement therapy (CRRT), the effect of convective (via continuous venovenous hemofiltration [CVVH]) versus diffusive (via continuous venovenous hemodialysis [CVVHD]) solute clearance on clinical outcomes is unclear. Our objective was to evaluate the feasibility of comparing these two modes in a randomized trial. ABSTRACT.METHODS: This was a multicenter open-label parallel-group pilot randomized trial of CVVH versus CVVHD. Using concealed allocation, we randomized critically ill adults with AKI and hemodynamic instability to CVVH or CVVHD, with a prescribed small solute clearance of 35 mL/kg/hour in both arms. The primary outcome was trial feasibility, defined by randomization of >25% of eligible patients, delivery of >75% of the prescribed CRRT dose, and follow-up of >95% of patients to 60 days. A secondary analysis using a mixed-effects model examined the impact of therapy on illness severity, defined by sequential organ failure assessment (SOFA) score, over the first week. ABSTRACT.RESULTS: We randomized 78 patients (mean age 61.5 years; 39% women; 23% with chronic kidney disease; 82% with sepsis). Baseline SOFA scores (mean 15.9, SD 3.2) were similar between groups. We recruited 55% of eligible patients, delivered >80% of the prescribed dose in each arm, and achieved 100% follow-up. SOFA tended to decline more over the first week in CVVH recipients (-0.8, 95% CI -2.1, +0.5) driven by a reduction in vasopressor requirements. Mortality (54% CVVH; 55% CVVHD) and dialysis dependence in survivors (24% CVVH; 19% CVVHD) at 60 days were similar. ABSTRACT.CONCLUSIONS: Our results suggest that a large trial comparing CVVH to CVVHD would be feasible. There is a trend toward improved vasopressor requirements among CVVH-treated patients over the first week of treatment. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov: NCT00675818 BODY.INTRODUCTION: Acute kidney injury (AKI) is a common complication of critical illness, afflicting up to two-thirds of patients admitted to the ICU [1]. A significant minority of patients with AKI requires renal replacement therapy (RRT), and these individuals have high short-term mortality that ranges from 50 to 70% [2]. In an attempt to mitigate these poor outcomes, various components of the RRT prescription have been rigorously examined in large well-designed randomized controlled trials (RCTs) [3-5]. The optimal mode of clearance in patients with AKI who require renal support is an area of considerable controversy resulting in significant practice variation [6]. Hemofiltration, whereby solutes are removed by convection, facilitates the removal of both low and higher molecular weight solutes, depending on the pore size of the membrane [7]. Hemodialysis, in which solute removal occurs via diffusion out of the bloodstream into the dialysate down a concentration gradient, removes low molecular weight molecules but provides limited clearance of higher molecular weight substances. When filter characteristics are kept constant, hemofiltration, which more faithfully mimics glomerular filtration, should result in the clearance of larger-sized solutes as compared to hemodialysis [7]. The removal of such solutes, which may include toxic mediators of sepsis and inflammation, provides the theoretical underpinnings for the superiority of hemofiltration as a renal support mode for critically ill patients with AKI. We conducted a multicenter pilot RCT of hemofiltration vs. hemodialysis in critically ill patients with AKI to determine whether a larger definitive trial based on clinically important endpoints would be feasible. In a secondary analysis, we evaluated whether hemofiltration improved global organ dysfunction. BODY.MATERIALS AND METHODS: BODY.STUDY DESIGN: We conducted an unblinded RCT of continuous venovenous hemofiltration (CVVH) vs. continuous venovenous hemodialysis (CVVHD) with concealed allocation (http://Clinicaltrials.gov registration number NCT00675818). Our reporting follows the updated CONSORT statement [8]. BODY.SETTING: Participants were recruited from ICUs at six academic hospitals: Mt. Sinai Hospital (Medical-Surgical ICU), Sunnybrook Health Sciences Centre (Critical Care Unit and Cardiovascular ICU) and St. Michael's Hospital (Medical-Surgical and Cardiovascular ICUs), all in Toronto, Canada; Victoria Hospital (Critical Care Trauma Centre), and University Hospital (Medical/Surgical Intensive Care Unit and Cardiac Surgery Recovery Unit), both in London, Canada, and University of Alberta Hospital (General Systems Intensive Care Unit) in Edmonton, Canada. The Research Ethics Boards of Mt. Sinai Hospital, St. Michael's Hospital, Sunnybrook Health Sciences Centre, London Health Sciences Centre and the University of Alberta approved the protocol. The Applied Health Research Centre at St. Michael's Hospital (Toronto, Ontario, Canada) was the trial coordinating center. BODY.POPULATION: We enrolled critically ill adults (≥ 16 years of age) with AKI, defined as a serum creatinine increase ≥ 50% from baseline (defined as the last known pre-morbid serum creatinine or earliest value available from the current admission). At the time of screening, at least one of the following indications for RRT initiation needed to be present: (i) oliguria (defined as urine output < 100 mL in the preceding 4 hours); (ii) metabolic acidosis (serum bicarbonate < 15 mmol/L and pH < 7.25); (iii) refractory hyperkalemia (serum potassium > 6 mmol/L despite medical efforts at potassium removal); (iv) serum urea > 50 mmol/L, or (v) suspected uremic organ involvement (pericarditis, encephalopathy, neuropathy or myopathy). Finally, participants needed to be hemodynamically unstable, defined as Sequential Organ Failure Assessment (SOFA)- Cardiovascular score ≥ 1 on the day of screening (see Additional file 1 for the modified SOFA score used in this study). This required the patient to have mean arterial pressure < 70 mmHg or receipt of at least one vasopressor or inotrope [9]. Patients were excluded if any one of the following was present: receipt of any RRT within the previous 2 months; presence of an obstructive etiology for AKI; receipt of a kidney transplant within the preceding year; diagnosis of rapidly progressive glomerulonephritis, vasculitis, or acute interstitial nephritis; a clinical indication for intermittent hemodialysis (for example, the presence of a dialyzable toxin); terminal illness with an associated life expectancy less than 2 months; moribund status (life expectancy < 48 hours as per judgment of physicians involved in the patient's care); prior enrolment in this study; enrolment in a competing ICU interventional study; non-availability of a CRRT machine, or administration of RRT for > 36 hours prior to eligibility assessment. BODY.PATIENT ALLOCATION: After eligibility was confirmed, we attempted to obtain consent from the patient or if the patient lacked capacity to consent, his/her substitute decision maker (SDM) was approached. A deferred consent option was approved at three sites, which allowed patient enrollment and randomization in the event of patient incapacity and the inability to locate an SDM. Using this mechanism, patients were randomized, and research personnel attempted to locate an SDM every 72 hours to affirm consent for participation. In all cases, when participants regained capacity, they were asked to provide consent if they were initially enrolled using a priori SDM consent or deferred consent; no participant withdrew consent once regaining capacity. Patients were allocated to a study group using sealed, opaque, sequentially numbered envelopes (prepared by the coordinating center) that were opened after consent was obtained [10]. Randomization was stratified by center in random blocks of four, six or eight. The Research Ethics Board at each center approved the study. An independent data and safety monitoring board tracked the trial's conduct. BODY.STUDY INTERVENTION: Participants randomized to CVVH were prescribed ultrafiltration with isovolemic replacement solution (evenly split between pre- and post-filter) to achieve a target clearance of 35 mL/kg body weight/hr. The prescribed hourly ultrafiltration rate was increased above 35 mL/kg/hr to compensate for the reduced efficiency of clearance related to the pre-filter component of the replacement solution volume administered. This adjusted CVVH dose was calculated from the post-filter replacement fluid (RF), pre-filter replacement fluid and blood (blood) flow rates as follows: Dose = Postfilter RF rate + ((Prefilter RF rate × (Blood flow/(Blood flow + Prefilter RF rate))). In the CVVHD arm, the dialysate flow was set to achieve a clearance of 35 mL/kg/hr, which included a post-filter hemofiltration flow of 100 to 200 mL/hr. This obligate low-volume post-filter hemofiltration is utilized at participating centers to minimize the risk of blood clotting in the machine's deaeration chamber. Ultrafiltration performed for achievement of net fluid balance or to compensate for the volume of administered anticoagulants (namely, citrate or unfractionated heparin) was not considered in the total dose. We used the patient's most recent measured body weight, as recorded in the chart or estimated by the study coordinator if no value was documented, to calculate prescribed RRT dose. Due to difficulties in achieving the target RRT dose as a result of high transmembrane pressures in some patients with higher body mass (particularly those randomized to CVVH), the protocol was modified in May 2010 (21 months after randomization of the first patient) such that the total fluid dose was capped at 4,000 mL/hr for both treatment arms, irrespective of the patient's weight. After this protocol change, twenty patients were enrolled in the trial; the 4,000 mL/hr dosing cap was invoked in six participants (five allocated to CVVHD and one to CVVH) who would have otherwise needed higher flows to achieve an actual total dose of 35 mL/kg/hr. All study therapies were delivered by the Gambro Prismaflex™ RRT machine using the ST100 (surface area 1.0 m2) or ST150 (surface area 1.5 m2) filter sets, which contain a polyacrylonitrile AN69 membrane (Gambro, Richmond Hill, ON, Canada). We permitted the use of any commercially available dialysate and replacement solutions. Decisions regarding circuit anticoagulation (heparin, regional citrate anticoagulation, or no anticoagulation) and volume control were at the discretion of the attending physicians. Patients remained on study therapy until death, withdrawal of CRRT as part of withdrawal of life support, hemodynamic stability (SOFA-cardiovascular score < 2 for > 24 hrs) permitting stepdown to intermittent hemodialysis, or recovery of kidney function (defined as urine output > 500 mL in the preceding 12 hrs, and most recent serum potassium < 5.5 mmol/L and serum bicarbonate > 18 mmol/L). BODY.OUTCOMES: The primary feasibility outcome of this study was the ability to administer > 75% of the prescribed CRRT dose to participants in each treatment arm. Secondary feasibility outcomes included the ability to enroll > 25% of fully eligible patients and the ability to follow > 95% of patients to 60 days following randomization (the anticipated follow-up period for the future definitive principal study). Secondary outcomes included change in SOFA score from baseline to days 1, 2 and 7, respectively, following randomization. Serial changes in SOFA scores have been shown to be correlated with clinical outcomes in critically ill patients with AKI who require RRT [11]. BODY.DATA COLLECTION: Trained research coordinators collected baseline clinical and demographic data, and information on pre-existing medical conditions. Specific risk factors for AKI were ascertained, including recent procedures, nephrotoxins, and sepsis (defined using consensus guidelines [12]). SOFA score was calculated at the time of randomization and on each day of study therapy. The SOFA-Cardiovascular score was modified to include the receipt of vasopressin. Patients receiving RRT on a given day were assigned a SOFA-Renal score of 4, regardless of urine output or serum creatinine. Participants were followed until death or a maximum of 60 days from randomization, at which time vital status and the ongoing need for RRT among survivors were recorded. BODY.STATISTICAL ANALYSES: As this was a feasibility trial with the primary objective of informing the design of a large-scale RCT, we planned to enroll a convenience sample of 75 participants from six sites. Patients who were randomized but never received RRT are described, but these individuals were replaced to ensure that at least 75 patients received some form of RRT. Since the primary feasibility outcome was based on the dose of CRRT received, patients for whom dose could not be readily calculated (those who received no RRT or forms of RRT other than CRRT) were excluded from the analysis related to feasibility. However, clinical outcomes are reported for all randomized participants. Descriptive statistics were used to characterize participants in either arm. Continuous variables are presented as means (SD) or medians (interquartile range, IQR) and two-group comparisons were performed with the t-test or Wilcoxon test, as appropriate. Two-group comparisons involving categorical variables were carried out with the chi square test. Analysis of covariance, adjusted for baseline SOFA score, was used to evaluate the change in SOFA score on days 1 and 2. Linear mixed models adjusted for baseline SOFA score and day of study therapy were used to evaluate the impact of RRT mode on SOFA score over the first week of therapy. For the fixed effect of treatment (that is, CVVH vs. CVVHD) 95% confidence intervals (CI) were obtained by profiling the log-likelihood function. All analyses were performed using R version 2.12.0 (R Development Core Team 2010, Vienna, Austria). BODY.RESULTS: We screened 347 patients; 143 were eligible for participation and 79 individuals (55.2%) were enrolled over a 24-month time period. The inability to obtain consent from the patient or SDM was the reason for the non-enrollment of otherwise eligible patients. One patient was excluded shortly after enrollment after it was decided to pursue a non-continuous form of RRT. In total, 78 patients were randomized (39 to CVVH, and 39 to CVVHD). In one case, prior to the start of therapy it was recognized that a patient randomized to CVVHD was inappropriately enrolled as the indication for RRT was toxin removal rather than AKI per se. This patient was excluded from all further analyses. Clinical outcomes are reported in an intention-to-treat fashion for the remaining 77 patients (CVVH, 39; CVVHD, 38). Four patients randomized to CVVH were excluded from the feasibility analysis, two due to death prior to commencement of study RRT, and two due to receipt of continuous venovenous hemodiafiltration as the initial mode of therapy. The indication(s) for RRT was (were) oliguria, metabolic acidosis, hyperkalemia and uremia in 34, 17, 6 and 8 patients, respectively, in the CVVH arm. In the CVVHD arm, these indications guided the inclusion of 36, 15, 4 and 3 participants, respectively. In total, 73 participants commenced the therapy to which they were randomized (CVVH, 35; CVVHD, 38); these individuals contributed to the analysis relating to the feasibility of treatment delivery (Figure 1). Figure 1Flow of patients through the trial. RRT, renal replacement therapy; CRRT, continuous renal replacement therapy; CVVH, continuous venovenous hemofiltration; CVVHD, continuous venovenous hemofiltration BODY.BASELINE CHARACTERISTICS (TABLE 1): The mean age of participants was 61.5 (SD 14.2) years and 30/77 (39%) were women. The median time from ICU admission to randomization was 2 (IQR 2 to 8) days. Sepsis was present in 63/77 (82%) of participants, 73/77 (95%) were mechanically ventilated and 69/77 (90%) received vasopressors at the time of randomization. Median 24-hr urine output was 125 (IQR 50 to 250) mL. Approximately two-thirds of participants received some form of RRT prior to randomization, with 23/39 in the CVVH group (59%) for a mean of 10.6 (SD 12.0) hrs, and 30/38 in the CVVHD group (79%) for a mean of 13.3 (SD 11.7) hrs. (Table 1 Table 1Baseline characteristics CVVH (n = 39) CVVHD (n = 38) Age, years 58.8 ± 13.7 64.3 ± 14.3 Female 14 (36%) 16 (42%) Weight, kg 86.6 ± 31.5 89.2 ± 26.4 Days from ICU admission to randomization 2 (1, 4) 2 (1, 3) SOFA score 16.3 ± 3.3 15.5 ± 3.0 Premorbid conditions Hypertension 19 (49%) 26 (68%) Diabetes mellitus 9 (23%) 9 (24%) Chronic kidney disease 5 (13%) 13 (34%) Acute kidney injury risk factors Sepsis 31 (79%) 32 (84%) Cardiopulmonary bypass in past 7 days 1 (3%) 4 (11%) IV contrast in past 7 days 9 (23%) 6 (16%) Physiologic parameters Urine output, mL/24 hr 125 (60, 245) 135 (46, 251) Minimum systolic BP during 24 hr before enrolment, mmHg 94.2 ± 16.5 86.1 ± 13.5 Minimum diastolic BP during 24 hr before enrolment, mmHg 48.9 ± 8.6 47.2 ± 9.4 Laboratory parameters Serum creatinine, µmol/L 276 (194, 352) 246 (155, 325) Urea, mmol/L 25.8 ± 38.1 17.6 ± 12.7 Potassium, mmol/L 4.3 ± 0.7 4.1 ± 0.6 Bicarbonate, mmol/L 20.5 ± 5.9 20.2 ± 5.3 Hemoglobin, g/L 86.1 ± 16.3 90.0 ± 15.2 Platelets, × 109/L 109.6 ± 82.6 127.2 ± 80.9 WBC, × 109/L 20.2 ± 43.3 16.9 ± 10.6 ICU interventions Duration of RRT prior to randomization, hours 8 (0, 19) 13 (1.0, 20.5) Mechanical ventilation 37 (95%) 36 (95%) Vasopressors 34 (87%) 35 (92%) Total parenteral nutrition 1 (3%) 2 (5%) Continuous data are displayed as mean ± SD or medians (interquartile range), as appropriate. Categorical variables are displayed as n (%). CRRT, continuous renal replacement therapy; CVVH, continuous venovenous hemofiltration; CVVHD, continuous venovenous hemodialysis; BP, blood pressure; RRT, renal replacement therapy; SOFA, Sequential Organ Failure Assessment; WBC, White blood cell. BODY.FEATURES OF STUDY TREATMENTS (TABLE 2): Among the 35 participants who started CVVH, RRT was prescribed for a median of 107 (IQR 55 to 146) hrs and delivered for 85 (IQR 43 to 128) hrs. Overall, 84.7% (95% CI 79.1, 90.3) of the prescribed duration was delivered. The mean weight-standardized dose was 33.6 (SD 7.4) mL/kg/hr while CRRT was ongoing.(Table 2 Table 2Feasibility and safety data CVVH(n = 35)CVVHD(n = 38)P-valueDuration RRT prescribed, hrs 146 ± 240 145 ± 156 0.88 Duration RRT received, hrs 130 ± 222 128 ± 142 0.87 Mean pre-filter replacement solution flow, mL/hr 1533 ± 442 0 n/a Mean post-filter replacement solution flow, mL/hr 1440 ± 488 180 ± 140 n/a Mean dialysate flow, mL/hr 0 2871 ± 872 n/a Mean RRT dose, mL/kg/hr 33.6 ± 7.4 34.7 ± 4.4 0.50 Prescribed dose delivered, % 84.7 ± 16.3 87.8 ± 13.7 0.73 Net ultrafiltration, L/day 1.7 ± 2.2 0.8 ± 4.1 0.98 Days on study therapy 5 (3-7) 4.50 (3.00-10.25) 0.79 Primary reason for CRRT withdrawal 0.75 Death while on CRRT 12 (35%) 10 (27%) Kidney function recovery 7 (21%) 7 (19%) Transfer to intermittent hemodialysis 12 (35%) 14 (38%) 3 (9%) 6 (16%) Catheter changes/day of therapy 0.09 ± 0.2 0.1 ± 0.2 0.92 Units RBCs transfused/day of therapy 0.3 ± 0.3 0.4 ± 0.7 0.38 Unscheduled circuit changes/day of therapy 0.2 ± 0.3 0.2 ± 0.2 0.36 Study days with receipt of norepinephrine, % 61.8 ± 36.2 65.7 ± 7.5 0.41 Study days with receipt of vasopressin, % 35.4 ± 35.9 43.0 ± 43.6 0.51 Continuous data are displayed as mean ± standard deviation or median (interquartile range), as appropriate. Categorical variables are displayed as n (%); n/a, not applicable. Data are restricted to patients who initiated therapy with the CRRT mode to which they had been allocated. RRT, renal replacement therapy; CRRT, continuous renal replacement therapy; RBCs, red blood cells; CVVH, continuous venovenous hemofiltration; CVVHD, continuous venovenous hemofiltration. CVVHD was prescribed in 38 participants for a median of 92 (IQR 57 to 145) hrs and delivered for 76 (IQR 44 to 148) hrs. Over the course of the trial, 87.8% (95% CI 83.3, 92.3) of the prescribed duration was delivered to patients randomized to receive CVVHD. The mean weight-standardized delivered RRT dose was 34.7 mL/kg/hr (SD 4.4). BODY.CLINICAL OUTCOMES: All subjects were followed to 60 days, by which point 22/39 (56%) and 21/38 (55%) of participants assigned to CVVH and CVVHD, respectively, had died. Among surviving patients, 4/17 (24%) and 3/17 (19%) of those initially assigned to CVVH and CVVHD respectively, were still dependent on RRT. After adjustment for baseline SOFA score, we found a non-significant decline in SOFA score among participants treated with CVVH compared to CVVHD on the first day (-0.4, 95% CI -1.3, 0.6) and second day (-0.4, 95% CI -1.6, 0.8) following randomization. Over the first week of therapy, the adjusted change in the SOFA score among participants treated with CVVH compared to CVVHD was -0.8 (95% CI -2.1, 0.5). The observed reduction appeared to be driven by a reduction in the cardiovascular component of the SOFA score (Figure 2). Figure 2Total and Cardiovascular (CV) Sequential Organ Failure Assessment (SOFA) scores over the first seven days following randomization. Total SOFA scores are denoted with squares; the cardiovascular component of the SOFA score is denoted with triangles.CVVH, continuous venovenous hemofiltration; CVVHD, continuous venovenous hemofiltration BODY.PROTOCOL VIOLATIONS AND ADVERSE EVENTS: We identified 15 protocol violations involving 15 participants. In one individual who started therapy with CVVH, achievement of the target dose was not feasible and RRT was supplemented with the addition of continuous dialysis; ongoing difficulties with maintaining adequate small molecule clearance using CRRT resulted in a change to sustained low efficiency dialysis. In five cases (one patient on CVVH, and four on CVVHD), stepdown from CRRT to IHD occurred before the participant met the study-defined criteria for hemodynamic stability. In five cases (two patients on CVVH, and three on CVVHD), RRT was discontinued altogether in participants who did not meet study criteria for renal recovery. No adverse events were attributed to the study intervention in either treatment arm. BODY.DISCUSSION: We completed a multi-center, concealed-allocation, randomized trial comparing hemofiltration and hemodialysis in critically ill patients with AKI. This pilot trial achieved its primary objective of confirming the feasibility of performing a large-scale trial evaluating RRT clearance mode in AKI. There was also a non-significant trend towards reduced organ dysfunction, driven by decreased vasopressor requirements, early after the initiation of RRT in patients who received hemofiltration. These feasibility data and the importance of the clinical question justify the conduct of a large trial with adequate power to evaluate the primary outcome of 60-day mortality. With a power of 0.80, type I error of 0.05, and a mortality reduction of 10% in patients treated with CVVH (estimated 60-day mortality in the CVVHD arm 55%), we estimate that such a trial would require the enrolment of nearly 400 patients per arm. A more conservative mortality reduction of 7.5% would require the enrolment of 700 patients per arm. Little is known about the optimal mode of clearance in renal replacement for AKI. Although both convection and diffusion remove small molecules with equal efficiency, convection may remove larger molecules that are not cleared by diffusive mechanisms [13]; previous studies have examined effects on inflammatory markers and have been too small to reliably determine effects on clinical outcomes. In addition, the profile of larger molecules removed by convection is relatively non-specific and may also include molecules that dampen inflammation or crucial medications such as antibiotics [14]. A randomized crossover study of 13 patients with AKI and the systemic inflammatory response syndrome found that CVVH for 24 hrs reduced plasma concentrations of TNFα and cleared more IL-6, compared to CVVHD. However, there was no effect on plasma concentrations of IL-6, IL-10, SL-selectin, or endotoxin [15]. Morgera and coworkers randomized 24 patients with sepsis-associated AKI to treatment with CVVH or CVVHD using a high cutoff membrane permeable to molecules up to 60 kilodaltons in size. Plasma concentration and clearance for IL-6 did not differ, but clearance of IL-1 receptor antagonist, an anti-inflammatory mediator, was enhanced by CVVH [16]. Of note, protein losses were higher in patients who received CVVH. In a prospective crossover study involving 15 patients with AKI who sequentially received CVVH and CVVHD, β2-microglobulin clearance was non-significantly higher among CVVH recipients (P = 0.055) [17]. Among trials focusing on clinical outcomes, a single-centre RCT (n = 20) did not demonstrate a difference in survival, renal recovery or ICU stay in patients treated with CVVH vs. CVVHD administered at fixed doses of 1.7 to 2.0 L/hr [18]. Similarly, a recent unpublished 65-patient RCT of CVVH vs continuous venovenous hemodiafiltration (both at 40 mL/kg/hr) did not demonstrate a survival difference at 28 days [19]. While interventional trials involving devices and processes of care that are susceptible to large variations in practice are challenging, we achieved our feasibility objectives. Specifically, we were able to recruit the majority of eligible subjects, implement the protocolized therapy for > 85% of the prescribed time and ascertain vital status at 60 days for all participants. When accounting for actual time on therapy, the delivered dose exceeded 80% of that prescribed in both treatment arms, thereby surpassing our feasibility threshold of 75%. Accordingly, we believe our study strongly supports the feasibility of a large definitive randomized trial comparing hemofiltration and hemodialysis in critically ill patients with severe AKI. This is the largest published trial to date to study the mode of solute clearance in AKI. Given the challenges of recruiting participants and implementing interventions in a population with a high burden of illness, the success of our pilot was a necessary precursor to a principal trial that examines patient-relevant clinical outcomes. Our eligibility criteria were pragmatic and assured the inclusion of individuals in North America who typically receive CRRT. A minority of individuals for whom no SDM could be found were enroled with deferred consent, thereby limiting exclusion of potentially eligible patients and mitigating selection bias. Of interest, no subject enroled by deferred consent had an SDM who subsequently withdrew consent or withdrew consent personally after regaining capacity. Finally, other than the clearance mode, all other aspects of RRT including stepdown to intermittent hemodialysis and withdrawal of RRT were performed in a manner consistent with usual practice. Our study has several limitations. As this was an unblinded trial, we cannot exclude the effect of co-interventions in either treatment arm. However, the nature of our intervention made blinding impractical, and we ensured that the two groups received equivalent RRT doses. There is also no definite intervention related to RRT prescription that has been shown to modify outcomes in critically ill patients with AKI. In addition, we cannot exclude the possibility that patients who were eligible but not randomized were systematically different than trial participants. Our protocol specified a target clearance of 35 mL/kg/hr, which was generally achieved in both arms. The decision to use this dose was guided by the fact that our trial was designed when higher dose CRRT was felt to be potentially beneficial based on data from two trials [20,21]. This pre-dated more recent trials and meta-analyses demonstrating no advantage of higher dose CRRT over doses of 20 to 25 mL/kg/hr, which would likely be the standard in a future trial comparing convection and diffusion [4,5,22,23]. However, since high dose therapy appears safe and may positively modify the effect of convective clearance [20], our higher dose target was reasonable. It should be noted that while 35 ml/kg/hr was prescribed to all participants, actual solute clearance may have been significantly lower due to changes in filter permeability [24,25]. On the other hand, when calculating the prescribed dose, we did not consider clearance associated with net ultrafiltration or removal of volume associated with the administration of anticoagulants. This approach would tend to underestimate the actual solute clearance that was delivered. Importantly, any deviation in dose from our 35 mL/kg/hr target would affect participants in both intervention arms to a similar extent. The Prismaflex CRRT system requires that all patients (even those predominantly getting hemodialysis) receive a small amount of post-filter hemofiltration (up to 200 ml/hr) to prevent clotting in the machine's deaeration chamber. In addition, CVVHD recipients would have had other unavoidable sources of convective clearance, specifically for the achievement of net fluid removal, and for the isovolemic removal of volume associated with the administration of the anticoagulant (citrate or heparin). Thus, CVVHD recipients did not receive purely diffusive solute clearance. We nonetheless estimate that the typical patient enrolled in the CVVHD arm still received > 80% of therapy in the form of diffusive clearance, which reassures us that this trial truly compared two different modes of solute clearance. Our trial was conducted using the AN69 polyacryonitrile filter, which has unique adsorptive characteristics [14]. While this is a widely used membrane in the administration of CRRT, the adsorptive characteristics of filters may differ and we cannot generalize our findings to settings in which other filters are utilized. Moreover, we did not collect data to evaluate the relative effect of CVVH or CVVHD on the removal of molecules of varying size. This trial was conducted using continuous RRT. Although the question of hemodialysis vs. hemofiltration is applicable to patients stable enough to receive intermittent RRT, machines to deliver intermittent hemofiltration were not widely available in North America when the trial was conducted. Among trial participants who became more hemodynamically stable, intermittent hemodialysis was utilized even when the initial CRRT mode was hemofiltration, thereby potentially diluting the benefits associated with CVVH. Finally, while we observed a trend towards improved organ failure scores, this finding must be interpreted with caution given the small sample size of our trial. Moreover, disease severity scores such as the SOFA score are surrogate markers that cannot supplant hard clinical endpoints. BODY.CONCLUSIONS: Our findings clearly support the feasibility of performing a definitive trial comparing CVVH and CVVHD in critically ill patients with AKI. The early non-significant trend towards reduced vasopressor requirements provides preliminary support to the concept that convective modes of clearance reduce inflammation and thus benefit critically ill patients. Given the high mortality associated with AKI, the lack of specific RRT interventions shown to reduce mortality, and current practice variation, the results of our pilot trial provide justification for a larger trial of hemofiltration vs. hemodialysis that will be adequately powered to evaluate meaningful clinical outcomes. BODY.KEY MESSAGES: • A randomized controlled trial of hemofiltration vs hemodialysis is feasible. • Hemofiltration may be associated with decreased vasopressor requirements over the first week of therapy. • A well-designed and adequately powered trial of hemofiltration vs hemodialysis would address an important area of uncertainty in the management of patients with AKI. BODY.ABBREVIATIONS: AKI: acute kidney injury; CRRT: continuous renal replacement therapy; CVVH: continuous venovenous hemofiltration; CVVHD: continuous venovenous hemodialysis; IL: interleukin; IQR: interquartile range; RF: replacement fluid; RCT: randomized controlled trial; RRT: renal replacement therapy; SDM: substitute decision maker; SOFA: Sequential Organ Failure Assessment; TNF α: tumor necrosis factor alpha. BODY.COMPETING INTERESTS: Dr. Bagshaw has served on a Gambro Experts Panel. No other relevant competing interests are reported. BODY.AUTHORS' CONTRIBUTIONS: RW conceived and designed the study, obtained funding, supervised study conduct, interpreted the data, drafted the manuscript, and provided critical review of the manuscript. JOF conceived and designed the study, supervised study conduct, interpreted the data, drafted the initial version of the manuscript, and provided critical review of the manuscript. SMB interpreted the study data, supervised study conduct, and provided critical review of the manuscript. KEB interpreted the study data, supervised study conduct, and provided critical review of the manuscript. AXG interpreted the study data, supervised study conduct, and provided critical review of the manuscript. MAH interpreted the study data, supervised study conduct, and provided critical review of the manuscript. AAH interpreted the study data, supervised study conduct, and provided critical review of the manuscript. SL interpreted the study data, supervised study conduct and provided critical review of the manuscript. DK interpreted the study data, supervised study conduct, and provided critical review of the manuscript. RMR interpreted the study data, supervised study conduct, and provided critical review of the manuscript. NIP interpreted the study data and provided critical review of the manuscript. KP participated in data analysis and interpretation, designed data collection tools, and participated in the critical review of the manuscript. KT participated in study design, conducted the data analysis, participated in data interpretation, and in the critical review of the manuscript. NKJA conceived and designed the study, supervised study conduct, interpreted the data, drafted the initial version of the manuscript, and provided critical review of the manuscript. All authors approved the final version. BODY.SUPPLEMENTARY MATERIAL: BODY.ADDITIONAL FILE 1: Modified Sequential Organ Failure Assessment (SOFA) score. SOFA score modified from the original version (see reference [9]) for application in the OMAKI trial. Click here for file

TITLE: Dental Amalgam Restorations and Children’s Neuropsychological Function: The New England Children’s Amalgam Trial ABSTRACT.BACKGROUND: A concern persists that children’s exposure to mercury vapor from dental amalgams produces neurotoxicity. ABSTRACT.OBJECTIVE: Our goal was to compare the neuropsychological function of children, without prior exposure to dental amalgam, whose caries were repaired using either dental amalgam or mercury-free composite materials. ABSTRACT.METHODS: We conducted a randomized controlled trial involving 534 6- to 10-year-old urban and rural children who were assessed yearly for 5 years using a battery of tests of intelligence, achievement, language, memory, learning, visual–spatial skills, verbal fluency, fine motor function, problem solving, attention, and executive function. ABSTRACT.RESULTS: Although the mean urinary mercury concentration was greater among children in the amalgam group than the composite group (0.9 vs. 0.6 μg/g creatinine), few significant differences were found between the test scores of children in the two groups. The differences found were inconsistent in direction. Analyses using two cumulative exposure indices—surface years of amalgam and urinary mercury concentration—produced similar results. ABSTRACT.CONCLUSIONS: Exposure to elemental mercury in amalgam at the levels experienced by the children who participated in the trial did not result in significant effects on neuropsychological function within the 5-year follow-up period. We previously reported on a randomized clinical trial, the New England Children’s Amalgam Trial (NECAT), in which no significant differences were found, over a 5-year follow-up interval, between the neuropsychological scores of children for whom dental amalgam was used to repair caries and the scores of children for whom mercury-free composite materials were used (Bellinger et al. 2006). The Full-Scale IQ score on the Wechsler Intelligence Scale for Children-Third Edition (WISC-III; Wechsler 1991) was the primary end point, and the General Memory Index (GMI) on the Wide Range Assessment of Memory and Learning (WRAML; Sheslow and Adams 1990), and the Visual Motor Composite (VMC) on the Wide Range Assessment of Visual–Motor Ability (WRAVMA; Adams and Sheslow 1995) were the two secondary end points. Each of these is a global score, derived by combining a child’s performance on tasks that assess somewhat different abilities. The additional analyses reported in this article address three issues. First, if mercury vapor liberated from dental amalgams produces specific rather than general neuropsychological effects and are most likely to be evident on tests that assess specific cognitive domains, global scores such as Full-Scale IQ, GMI, and VMC might be relatively insensitive to important treatment-group differences. Even in the absence of treatment-group differences on global test scores, differences in specific cognitive domains could, depending on their nature and severity, represent morbidities with important consequences for children’s health and well-being. Therefore, here we report comparisons of the scores of the amalgam and composite groups on the sub-scales that contribute to Full-Scale IQ, the GMI, and the VMC, as well as scores on a battery of additional, domain-focused, neuropsychological and educational tests. Second, the exposure index used in the primary analyses of the trial was treatment-group assignment. This could have introduced a form of exposure misclassification insofar as the variability in the treatment needs of the children in the amalgam group resulted in the receipt of variable amounts of amalgam and thus in their potential exposure to mercury. Therefore, we repeated the analyses replacing treatment-group assignment with two continuously distributed indices of exposure: surface-years of amalgam and urinary mercury concentration. Third, it is possible that only a subset of children experienced adverse neuropsychological effects as the result of exposure to amalgam, either because of behaviors, such as bruxism or frequent gum chewing, that cause enhanced release of mercury (Barregard 2005; Barregard et al. 1995) or because of enhanced sensitivity to mercury. Two recent studies of dental professionals suggest that polymorphisms for brain-derived neurotrophic factor (Echeverria et al. 2005; Heyer et al. 2004) and the coproporphyrinogen oxidase gene (Echeverria et al. 2006) modify the neurotoxicity of elemental mercury. If the prevalence of such enhanced vulnerability to elemental mercury is low or the associated increase in neuropsychological toxicity is modest in magnitude, its impact on the mean scores of children in the amalgam group might not have been sufficiently large to produce significant treatment-group differences. In an attempt to identify the presence of a subgroup of children who are particularly sensitive to amalgam, we compared the distributional characteristics of the scores within the treatment groups. BODY.METHODS: BODY.STUDY DESIGN AND PARTICIPANTS: Children were eligible if they were 6–10 years of age, fluent in English, had no known prior or existing amalgam restorations, had two or more posterior teeth with dental caries, and did not have a physician-diagnosed psychological, behavioral, neurological, immunosuppressive, or renal disorder (Children’s Amalgam Trial Study Group 2003). Children were recruited from several community dental clinics in the Boston/Cambridge area of Massachusetts, an urban setting, and from a dental clinic in Farmington, Maine, a rural area. A total of 5,116 children were screened for eligibility. Eligibility was confirmed for 598 children. At baseline visits, children received a dental examination by a study dentist, X rays, and standard preventive dental care (e.g., cleaning, application of sealants). Other baseline visit activities included the collection of blood and urine samples, anthropometric measurements of height, weight, and body fat, neuropsychological testing of the child, a health interview with the child’s guardian, and neuropsychological testing of the guardian. After completion of baseline visits, children were randomized to a study treatment arm. Randomization was stratified by geographic location (Boston/Cambridge vs. Farmington) and number of teeth with caries (two to four vs. five or more), using randomly permuted blocks within each of the four strata. The NECAT was conducted in accordance with all applicable requirements for the protection of human subjects. All children provided assent and parents provided informed consent. The study protocol was approved by the institutional review boards of the New England Research Institutes, the Forsyth Institute, and the clinics from which children were recruited. BODY.INTERVENTIONS AND FOLLOW-UP: All children had semiannual dental examinations as well as additional visits required to meet treatment needs. For children in the amalgam arm, a dispersed-phase amalgam was used to restore all posterior teeth with caries at baseline and to repair incident caries during the 5-year trial period. For children in the composite arm, composite material (white filling) was used for all restorations. Following standard clinical practice, for both groups, composite material was used to repair caries in the front teeth, and stainless steel crowns were used to restore primary teeth with extensive lesions that could not otherwise be restored. The choices of dental materials and techniques were standardized across sites and dentists. Urine samples were collected annually and analyzed for elemental mercury using cold vapor atomic absorption. Values were expressed as micrograms per gram creatinine. The analyses reported use only urinary mercury concentrations in samples collected at 3, 4, and 5 years of follow-up. After 1 February 2000, the detection limit, which had been 1.5 ng/mL, was reduced to 0.45 ng/mL as a result of increasing the volume of sample analyzed for each child. Samples with a mercury concentration below the detection limit were assigned a value of 0.45/√2 (Bellinger et al. 2006). Participants and dentists could not be blinded to treatment assignment, but all individuals who collected outcome data (e.g., neuropsychological tests) or analyzed specimens (e.g., for mercury) were blinded to children’s treatment assignments. BODY.NEUROPSYCHOLOGICAL ASSESSMENTS: At baseline, before randomization and the receipt of any dental treatments, children participated in two 3-hr neuropsychological test sessions. At the first session, the WISC-III (Wechsler 1991) and the Wechsler Individual Achievement Test (WIAT) (Psychological Corporation 1992) were administered. These tests were again administered at 3 and 5 years after baseline. The second baseline session consisted of a battery of domain-focused tests. This test battery, which was again administered at 1, 2, and 4 years after baseline, included the WRAML (Sheslow and Adams 1990), the WRAVMA (Adams and Sheslow 1995), the Trail-Making Test (Spreen and Strauss 1991), finger tapping (the WPS Electronic Tapping Test; Western Psychological Services, Los Angeles, CA), ordered and unordered verbal cancellation (Mesulam 1985), category fluency (McCarthy 1972), the Controlled Oral Word Association Test (letter fluency) (Spreen and Strauss 1991), simple visual reaction time (the Standard Reaction Timer; Software Science, Cincinnati, OH), the Stroop Color–Word Interference Test (Trenerry et al. 1989), and the Wisconsin Card Sorting Test (Heaton et al. 1993). A total of 14 testers were used at the Boston/Cambridge site and five testers at the Farmington site. Quality control of the assessments was assured by having all examiners trained and certified by one supervising psychologist (D.C.B.) before conducting assessments of children enrolled in the trial, and monitored over the course of data collection. Each testing session was completely rescored by a second individual and errors were corrected. A variety of computerized algorithms were used to check the entered data for internal consistency. BODY.SAMPLE SIZE DETERMINATION: The trial was designed to achieve 80% power to detect a 3-point difference between treatment arms in 5-year change in Full-Scale IQ score, adjusted for baseline IQ score and randomization stratum. Assuming a retention rate of 75% over the 5-year follow-up period, the recruitment goal was 250 children per treatment arm, for a total sample size of 500 children. BODY.STATISTICAL ANALYSIS: We used intention-to-treat analyses, using analysis of covariance, to compare the amalgam and composite groups in terms of the changes, over 5 years, in scores on the WISC-III and WIAT and the changes, over 4 years, in scores on the domain-focused tests. These analyses thus indicated whether the central tendencies of the distributions of change scores differed in the amalgam and composite groups. Adjustments were made for baseline covariates: test score, randomization stratum, age, sex, socioeconomic status, hair mercury, and blood lead level. We calculated socioeconomic status using the method developed by Green (1970). Hair mercury was included to control for methylmercury, a form of mercury that is known to be a developmental neurotoxicant (Weiss 2006) but acquired primarily by consumption of contaminated seafood. Elevated blood lead level is a well-known developmental risk factor, with an increased prevalence among children who are socioeconomically disadvantaged (Bellinger 2006). We evaluated children’s neuropsychological test scores in relation to two continuously distributed indices of exposure. The first index was surface-years of amalgam (number of amalgam surfaces weighted by number of years present), calculated from the information contained in dental clinic records regarding dates of amalgam placement, the number of tooth surfaces involved in the restoration, the timing of loss of primary teeth containing amalgam restorations, and the like. The second index was urinary mercury concentration. Scores on the WISC-III and WIAT, both of which were administered at year 5 of follow-up, were evaluated in relation to the mean of available urinary mercury concentrations at years 3, 4, and 5. Scores on the other tests, which were administered for the final time at year 4 of follow-up, were evaluated in relation to the mean of available urinary mercury concentrations at years 3 and 4 of follow-up. We evaluated the associations between these indices of exposure and children’s neuropsychological test scores using analysis of covariance, adjusting for the same set of baseline covariates used in the intention-to-treat analyses. We conducted the analyses using the Kolmogorov-Smirnov test (Stuart and Ord 1991) to compare characteristics other than the central tendency of the distributions of the change scores in the treatment groups. When the results indicated that the change scores of children in the two groups did not come from the same distribution, we determined whether this was attributed to differences between treatment groups in the percentages of children with change scores indicating substantial deterioration of performance over the follow-up interval. Of particular interest was whether, in the absence of a treatment-group difference in mean change score, a greater percentage of children in the amalgam group than in the composite group demonstrated such deterioration. To evaluate the impact of interexaminer variability on the results, we repeated the intention-to-treat analyses including adjustment for a set of indicator variables representing the neuropsychological examiners. BODY.RESULTS: Table 1 shows that the treatment groups did not differ significantly in terms of age, race, household income, education of primary caregiver, Full-Scale IQ, hair and urinary mercury concentrations, blood lead level, and number of decayed tooth surfaces. Females outnumbered males in the composite arm. Children were primarily non-Hispanic white (62%), with non-Hispanic blacks making up an additional 19% of the sample. The mean number of total decayed tooth surfaces at baseline was 9.5, with 1.7 of the surfaces being in permanent teeth. Slightly more than half of the children (54%) had five or more teeth with caries that required restoration, with the rest having two to four carious teeth. Children from the Boston/Cambridge area tended to have more caries than did children from Farmington (10.3 vs. 8.6 carious surfaces, respectively). Children continued to have dental treatment needs over the course of the 5-year follow-up period, averaging approximately one new filled surface per year. The treatment needs were similar in the treatment groups (Table 2). At the end of the follow-up period, the number of restored surfaces in place ranged from 0 to 36 in both treatment groups, and the mean values did not differ significantly (p = 0.16). The amalgam and composite groups also did not differ significantly in the cumulative number of restored surfaces over the trial (p = 0.10). In the amalgam group, 79% of the surfaces had been restored with amalgam. At the end of the 5-year follow-up period, the mean urinary mercury concentration was significantly greater among children in the amalgam group (0.9; range, 0.1–5.7 μg/g creatinine) than among children in the composite group (0.6; range, 0.1–2.9 μg/g creatinine) (p < 0.001). Table 3 shows the change scores over the follow-up interval for each test score. Of all the change scores evaluated, only two differed significantly between the amalgam and composite treatment groups. On the Number–Letter Memory subtest of the WRAML, the 4-year change score of the amalgam group was significantly more positive than was the change score of the composite group, indicating greater improvement over time in the amalgam group. The 4-year change in the time required to complete Part B of the Trail-Making Test was significantly more negative in the composite group than in the amalgam group, indicating greater improvement over time in the composite group. The results of analyses using surface-years of amalgam or urinary mercury concentration as the exposure metric were consistent with those of the intention-to-treat analyses, providing no evidence of a detrimental effect of amalgam on children’s test scores. The coefficient for surface-years of amalgam was significant for three scores (Picture Memory and Number–Letter Memory of the WRAML and letter fluency), but for all three scores, the sign was positive, indicating that the score improved with increasing exposure to amalgam (Table 4). Urinary mercury concentration was not significantly associated with any of the test scores (Table 5). The results of the Kolmogorov-Smirnov tests indicated that the only two scores for which the distributions of changes scores in the amalgam and composite groups differed significantly were two subtests of the WRAML: Finger Windows and Number–Letter Memory (Table 3). On both tests, however, children in the amalgam group showed greater improvement over time than the children in the composite group, with the difference being significant for Number–Letter Memory. The distributions of change scores were not significantly different on the Trail-Making Test Part B (time to complete), the test on which the composite group showed significantly more improvement than the amalgam group. Adjustment for neuropsychological examiner did not produce results that were appreciably different (data not shown). BODY.DISCUSSION: These analyses revealed an absence of consistent differences between the scores of children in the amalgam and composite treatment groups on a battery of neuropsychological tests that assessed a wide range of domains, including intelligence, achievement, language, memory, learning, visual–spatial skills, verbal fluency, fine motor function, problem solving, attention, and executive function. The findings were similar when the dichotomous variable treatment-group assignment was replaced by two continuously distributed indices of exposure, one that combined the amount and duration of amalgam a child received and one that was a biomarker, urinary mercury concentration. Furthermore, no evidence was found to support the hypothesis that a subset of children in the amalgam group suffered substantial harm. The number of significant differences observed was similar to that which might have been expected to occur by chance. Although neuropsychological deficits associated with amalgam exposure have been reported in several studies of dental professionals (Bittner et al. 1998; Echeverria et al. 1995, 1998; Ngim et al. 1992) and others exposed occupationally to mercury (Rohling and Demakis 2006), our findings are similar to those involving mercury exposure in cohorts drawn from the general population of adults and children (Brownawell et al. 2005). In a cross-sectional study of 550 30- to 49-year-old healthy employed adults, scores on tests of verbal memory, nonverbal memory, attention, psychomotor speed, and fine motor coordination were not significantly associated with any of several exposure indices considered (number of visible amalgam surfaces, number of visible occlusal amalgam surfaces, urinary mercury concentration) (Factor-Litvak et al. 2003). The mean urinary mercury concentration in that cohort of adults, 1.7 μg/g creatinine, was higher than the mean concentration of 0.9 μg/g creatinine among the children in the amalgam treatment group in our trial 5 years after placement of their first amalgam restorations. In a study of 1,663 Vietnam-era veterans, the total number of tooth surfaces with amalgam fillings was unrelated to clinical neurological signs (e.g., tremor, coordination, station, gait, strength, sensation, muscle stretch reflexes, or indices of peripheral neuropathy), although it was associated with vibrotacile sensation in non-diabetic participants (Kingman et al. 2005). In a study of 384 German 6-year-olds, 24-hr urinary excretion of mercury, which averaged 0.16 μg, was not significantly related to scores on a variety of tests, including the Vocabulary and Block Design subtests of the WISC and five tests of the computerized Neurobehavioral Evaluation System 2 (pattern comparison, pattern memory, tapping, simple reaction time, continuous performance test) (Walkowiak et al. 1998). In this cohort, some indices of visual contrast sensitivity did decline with increasing urinary mercury excretion, however (Altman et al. 1998). Over the course of the follow-up interval, the scores of children in both treatment groups tended to change in the direction of improved performance, even on tests for which scores are standardized for age. Several factors might have contributed to improved performance over time. First, this could represent a type of sampling bias, reflecting the characteristics of families who are motivated to enroll in such a trial and to participate for its full duration. Second, all tests except the WISC-III and WIAT were administered yearly, so the general improvement in scores might reflect the familiarity that children developed with the test materials and expectations. Particularly large improvements tended to be on performance-based tests, such as the WRAVMA pegboard and the Processing Speed composite of the WISC-III, one component of which is Symbol Search, a timed task that involves matching symbols and digits. A substantial improvement was also noted on the WRAML Learning Index, which reflects the rapidity with which a child learns new material, such as sound–symbol pairs, a word list, and the locations of hidden designs. Repeated administration of these tasks, even at yearly intervals, might be expected to result in an increased rate of acquisition of the material. As noted, the dental treatment needs of the children enrolled in the trial were substantial and exceeded those typical of the general population of U.S. children. For example, among 6- to 11-year-old children who participated in the National Health and Nutrition Examination Survey (NHANES) 1999–2002, the prevalence of dental caries in primary teeth was 22%, and the mean numbers of decayed or filled primary teeth and surfaces were 1.7 and 3.7, respectively (Beltran-Aguilar et al. 2005). The prevalence of dental caries in permanent teeth was 20% (mean number of decayed, missing, and filled teeth and surfaces in permanent teeth were 0.1 and 0.4, respectively) (Beltran-Aguilar et al. 2005). Therefore, children assigned to the amalgam group in the NECAT are likely to have experienced greater exposures to mercury vapor from amalgams than do most children in the United States. Given our failure to detect significant differences between the amalgam and composite groups in neuropsychological function, these results provide reassurance that the use of dental amalgam to repair caries is not producing substantial neuropsychological morbidity in the general population of children in the United States. The conclusions must be tempered, first, by a recognition that the follow-up interval of 4–5 years might have been too short to allow for the expression of such deficits. Second, the critical window of children’s greatest vulnerability to elemental mercury might already have passed by the time the children were enrolled in the trial (≥ 6 years of age). Given the heightened sensitivity of the fetus to methylmercury, prenatal exposure to mercury vapor, which is known to cross the placenta, warrants increased attention. In the NHANES 1999–2000 survey, among women of child-bearing age, an increase of 10 dental surfaces restored with amalgam was associated with an estimated increase of 1.8 μg/L in urinary mercury concentration (Dye et al. 2005). Mercury level in amniotic fluid is weakly associated with number of amalgam fillings (Luglie et al. 2005). A recent case–control study did not, however, find an increased risk of delivering a low-birth-weight infant among women who had up to 11 amalgam restorations placed during pregnancy (Hujoel et al. 2005). The results of studies of the reproductive outcomes of women with dental workplace exposures have been mixed (Dahl et al. 1999; Elghany et al. 1997; Ericson and Kallen 1989). In some of these studies, distinguishing the potential impact of mercury exposure from the impacts of other workplace exposures, such as to disinfectants containing ethanol and benzene, is difficult. Third, the prevalence of children with enhanced sensitivity to elemental mercury might be too low among the children enrolled in the NECAT for us to have

TITLE: Effects of Guideline-Based Training on the Quality of Formal Ontologies: A Randomized Controlled TrialGuideline-Based Training on Quality of Ontologies ABSTRACT.BACKGROUND: The importance of ontologies in the biomedical domain is generally recognized. However, their quality is often too poor for large-scale use in critical applications, at least partially due to insufficient training of ontology developers. ABSTRACT.OBJECTIVE: To show the efficacy of guideline-based ontology development training on the performance of ontology developers. The hypothesis was that students who received training on top-level ontologies and design patterns perform better than those who only received training in the basic principles of formal ontology engineering. ABSTRACT.METHODS: A curriculum was implemented based on a guideline for ontology design. A randomized controlled trial on the efficacy of this curriculum was performed with 24 students from bioinformatics and related fields. After joint training on the fundamentals of ontology development the students were randomly allocated to two groups. During the intervention, each group received training on different topics in ontology development. In the assessment phase, all students were asked to solve modeling problems on topics taught differentially in the intervention phase. Primary outcome was the similarity of the students’ ontology artefacts compared with gold standard ontologies developed by the authors before the experiment; secondary outcome was the intra-group similarity of group members’ ontologies. ABSTRACT.RESULTS: The experiment showed no significant effect of the guideline-based training on the performance of ontology developers (a) the ontologies developed after specific training were only slightly but not significantly closer to the gold standard ontologies than the ontologies developed without prior specific training; (b) although significant differences for certain ontologies were detected, the intra-group similarity was not consistently influenced in one direction by the differential training. ABSTRACT.CONCLUSION: Methodologically limited, this study cannot be interpreted as a general failure of a guideline-based approach to ontology development. Further research is needed to increase insight into whether specific development guidelines and practices in ontology design are effective. BODY.INTRODUCTION: Formal ontologies are software artefacts that represent theories which attempt to give precise mathematical formulations of the properties and relations of certain entities [1]. In the biomedical domain, formal ontologies have been promoted as a key resource to enable knowledge management. Typical applications are semantic annotations of experimental data (e.g. high-throughput data from bioassays), information extraction applied to clinical documents, as well as the integration of heterogeneous databases and services [2]–[5]. A large number of biomedical ontologies have been created for a wide range of subject matters, and many of them have successfully been used, particularly in genetics and proteomics [6], [7]. In the meantime the support of formal ontology as an engineering discipline has been consolidated, and repositories for the standardized access to ontologies have been made available [8]–[11]. However, the use and re-use of ontologies is often limited due to insufficient representational and formal quality. A variety of errors in ontologies and their consequences have been described [12]–[18]. Principal causes for these problems are the complexity of ontology engineering together with the limited availability of trained ontology developers. A skilled ontology developer should have practical knowledge in four key areas. Potentially, the most important knowledge source required for successful ontology engineering is the domain itself which is to be represented. Without a deep and sound understanding of the domain entities and the relations between them, ontology developers have nothing they can even start with. For instance, they will just fail when they have to face the task of building a correct taxonomic hierarchy of classes of things in an unknown domain, even before it comes to the design of more complex structures like partonomies, i.e. models that relate parts and wholes. Domain entities like objects, processes, qualities, functions etc., together with the relations that connect them can only be represented after acquiring a thorough understanding of the domain. Even though, there is a considerable risk to design formally correct ontologies which however miss the very point the ontology developer intended to be represented, thus ending up in a bad representation of reality. The second requirement for successful ontology development is the mastery of the formalism used, i.e. the logical expression language, with detailed knowledge on how the language is composed (syntax) and what the expressions in this language mean (semantics). The formality and rigidity of logics is one of the largest obstacles for developers that come from their own domain, without having a mathematics or philosophy background. Experienced domain expert generally have no difficulty to express their knowledge in precise textual statements. However, human language allows for subtle distinction to express the whole range from universal truths to contingent facts. Formal ontologies, however, are restricted to formulate what is universally true in a domain. Many pieces of domain knowledge are outside the scope of formal ontology. To recognize this limitation requires training and experience. Furthermore, human language is misleading. Often, even basic syntactic constructs like head nouns with adjectival modifiers require diverging interpretations: a complicated pregnancy is a pregnancy, but a suspected pregnancy isn’t. Nevertheless we have seen that even experienced ontology developers are influenced by the idiosyncrasies of natural language and introduce inadequate meaning to formal constructs. Visual tools like the Protégé Ontology Editor allow to hide the bare formalism of logic-based description languages like the Web Ontology Language (OWL) from the developer. However, the ontology developer should be fluent in using the corresponding editor functions and know the meaning of the constructs of the formal language. On top of the representational language, an ontology developer should be able to build on existing specifications and standards. A large body of consolidated frameworks on how domain entities should be categorized and represented is provided by various disciplines like philosophy, mathematics and information sciences, in particular top-level ontologies and ontology design patterns (ODPs). Top-level ontologies, like BFO, DOLCE or BioTop provide a foundation of foundational categories (e.g. process, material object, quality) and relations (e.g. part-of, participates-in), to be extended and specialized by domain ontologies. Ontology design patterns are inspired by so called software design patterns originating in software technology [19]–[20] forming complex elements of software code to be re-used in development scenarios with similar requirements or functionality. Both a top-level ontology and ontology design patterns provide re-usable building blocks to guide the development of new domain ontologies and to sustain ontology standardization and interoperability. The development of an ontology is often driven by its later application. Here, the developer should understand the technical framework in which the ontology is to be embedded. This can range from the support of natural language processing to interoperability frameworks and visual navigation tools. For each application scenario, different features of an ontology might be important. While the segment of reality represented in an ontology remains constant across applications, the scope, the depth and the implementation of the representation is influenced by the use-case. While the core knowledge necessary to develop ontologies originates in the domain to be represented, domain knowledge has to be connected with knowledge about the semantics of the representational languages and the ontological framework in which the representation is grounded, i.e. basic categories, relations, and constraints. Therefore domain experts who develop ontologies should stand on a solid ground in computer science, logics, and philosophy, either by teaming up with experts from these disciplines or by acquiring related skills and knowledge by targeted training. Guided by this rationale, we have developed a guideline on good ontology design. It comprises what we consider to be the most essential knowledge to develop good quality ontologies (GoodOD guideline (http://purl.org/goodod/guideline)) using description logic (DL) for representation and reasoning. This guideline combines foundations from philosophical ontology and logics, with the representational language OWL DL, and is rooted in the domain top-level ontology BioTop [21]. The guideline itself is not intended as training material for beginners in the field of ontology engineering but as a concise report which can be used for consultation on the constituents of good ontology development. To actually provide domain experts with the knowledge from the GoodOD guideline, we implemented it in a curriculum [22], [23]. The aim of this study was to provide evidence that guideline-based training of ontology developers enhances their performance. Therefore, the effect of training of a certain problem solution based on our guideline was tested against the effect of training in ontology design that was not specific for this problem. This was done as a randomized controlled trial with students from the biomedical domain with a background in computer sciences. A set of ontology similarity (distance) metrics for ontologies was used as a measurement instrument. As primary outcome measure, similarity metrics were applied to compare the ontology artefacts created by students with a gold standard artefact created by experts. As a secondary outcome measure the intra-group ontology similarity was compared between groups. We assumed that an improved quality would manifest itself in two ways: (i) as a higher similarity of trained students’ artefacts to expert ontology artefacts, and (ii) in a larger similarity between products of trained students compared to products of untrained students. BODY.QUALITY OF ONTOLOGIES IN THE BIOMEDICAL DOMAIN: Ontologies use logic to formulate statements about what experts consensually assumed to hold true in a domain, with scope and granularity varying according to the use cases for which the ontologies are created. To evaluate their quality is inherently difficult, as it depends on the definition of quality, as well as on the availability of measurement instruments (see below). The literature about quality problems in domain ontologies is rich, but difficult to be operationalized. Design errors described in the literature can be categorized into different types of various complexities: The violation of implicit or explicit naming conventions [24] result in lexical names of classes or relations which are likely to be misinterpreted or easily to be confused by humans. Other errors arise through the mix-up of commonsense meaning of natural language expressions with the semantics of the representational DL language. E.g., on the background of natural language pragmatics a value restriction like ‘OxygenMolecule hasPart only OxygenAtom’ can be wrongly understood to imply the existence of parts when seen throuth the lens of natural language pragmatics [18]. However, as a DL expression, a value restriction only limits the possible target classes of a relation without implying the existence of a relatum, i.e. something that is related. Hence, our sample statement says that the parts can only be oxygen atoms (which is wrong, as oxygen molecules also have protons as parts), without stating that every oxygen molecule consists of oxygen atoms (which is true but needs a different logical statement). Other failures are due to problems with the counterintuitive semantics of DL. We are used to understand that the absence of a statement on a fact does imply its negation. This is the commonsense interpretation throughout many applications of daily life and even in science. In description logic the absence of a statement from an ontology, i.e. an axiom, does not imply its negation (the so called open world assumption) with all its consequential implications. A very common error is the absence of explicit statements that things cannot be in the same class but are in disjoint classes: mutually disjoint partitions are not made explicit [3]. For instance, if an ontology developer creates the two sibling classes Animal and Plant, nothing is said about whether things can exist that are plants and animals at the same time. Although or just because of the small number of language elements and its consequential restricted expressivity, ontologies based on DL can contain a multitude of logical inconsistencies [25]. A large and important class of errors is related to inadequate representations of the underlying reality itself that do not surface as inconsistencies or contradictions in a logical sense. They are mostly due to an incomplete or wrong understanding of the domain which is to be represented in the ontology. Consequently, they result in underspecification and inaccuracy regarding the underlying ontological premises. These errors are difficult to detect because the ontology reveals no logical contradictions when checked by a DL classifier. Only domain experts with a sound ontological background can detect these errors. They are able to understand the meaning of the critical representation as wrong underlying assumptions. In many cases, faulty ontologies were built without making the underlying assumptions explicit.However, when there are competing or incongruent ontological commitments in the background, they can be even more problematic [26], [27]. Not only the erroneous axioms themselves, but (potentially even worse) also their computed entailments have impact on basic tasks like equivalence detection, ontology alignment, and affect interoperability in general. Above all, ontologies are often not easily applicable in usage scenarios for which they have not been developed. An essential condition for the overall success of an ontology is that it can be used across a variety of use-cases and applications, and that it can seamlessly interact with other semantic resources. Recently, some activities have emerged that target assurance and improvement of ontology quality. Coordination efforts from policy and best practice providers [8] assure the quality of ontologies provided by repositories [9], [10] and tools support the detection and correction of typical errors [17], [24]. BODY.TRAINING IN FORMAL ONTOLOGY: A variety of educational material is available on ontology, logics and knowledge representation. It is presented in different forms like textbooks, tutorials, online courses, and lectures and has been written to facilitate ontology engineering for a broad range of learners from different domains. This material is mostly technically oriented and focuses on certain skills, e.g. the usage of Protégé editor or how to represent a specific domain for a specific usage scenario. A public repository especially of training material dedicated to OWL and bio-medical ontology is available at the website of the CO-ODE project (http://www.co-ode.org). Among introductory and expert material on the development of ontologies with the famous Protégé-OWL tutorial is linked using a Pizza-ontology as an example [28]. Sources for training material on ontology theory and ontology engineering are websites of individual researchers, most prominently the website of Barry Smith at the National Center for Ontology Research (NCOR, http://ontology.buffalo.edu/smith/). At the National Center for Biomedical Ontology (NCBO) some audio-visual material is available esp. on the usage of BioPortal [9]. It is not possible to mention and evaluate all available educational material on ontology and ontology development here. Most of the material is of high quality and is the result of long term educational efforts, which cannot be valued high enough. But many of these resources are focused on only one aspect of ontology, e.g. describing the expressivity of representational languages in the context of editing and reasoning tools, typically description logics with the Protégé editor and plugin reasoners. Other materials tend to be agnostic of the principles we formulated above like the use of existing upper-level ontologies and design patterns. The curriculum we evaluated in this study integrated everything we considered to be necessary for successful ontology development beyond the knowledge of the domain itself. It is targeted especially to those we consider the most important future ontology developers, viz. students with a domain background in the life sciences. Furthermore, the curriculum is based upon an explicit guideline on good ontology development which we believe provides a necessary, consistent and sufficient core definition of good ontology design. All modules of the curriculum are built up on each other and use consecutive exercises and examples from the same domain ontology. BODY.ONTOLOGY EVALUATION: There are numerous accounts of the evaluation of ontology artefacts. However, objectives, outcome measures and evaluation methods differ on a large scale, as they need to be aligned to the variant semantic flavours and expressivity, which in turn depend on the ultimately envisioned use case scenarios. Some quantitative metrics and frameworks for quality measurement of DL ontologies are available [29]–[31]. All of them are highly synthetic and take into account features of the ontology that – prima facie – seem to be correlated with its quality (such as connectedness, depth of the hierarchy, annotation coverage, etc.). These metrics are often fraught with the problem that they are not validated independently, a fact that is largely due to the unavailability of a generally agreed upon and easy to operationalize account of ‘ontology quality’. Hence no simple objectified quality assessment method has emerged so far. As a consequence, inter-rater reliability between human experts assessing the ontologies is often the method of choice for determining ontology quality [18], though it is infeasible for larger quantities of ontology content. As mentioned, the perceived quality characteristics of an ontology are very often dependent on the use case they were designed for. Some authors conclude that “[r]esearch results in biomedical ontology should always be evaluated against a biomedical task for which the ontologies are intended” [32]. This places ontology engineering in a light quite similar to software engineering, and puts the focus of ontology quality assurance on areas similar to quality assurance for software artefacts, which is also reflected in the adaptation of the SQuaRE quality model [31], [33]. We agree that usability is a key criterion for the successful adoption of ontologies in real world applications and firmly believe such approaches to be generally laudable since they try to address the needs of ontology users. Nonetheless, an exclusive focus on usability and use-case oriented quality criteria may fall short of capturing the fact that – to some extent – ontologies are more like standards than like software artefacts. In some areas of ontology research, these problems are less pressing. For example, the quality of ontologies generated automatically by machine learning or text mining procedures or of automatically derived ontology alignments [34], [35] can be readily assessed by their similarity to a manually curated gold standard which is normally done, e.g., in competitions for text mining algorithms. To our knowledge there are no prior studies that apply this approach to the assessment of ontology artefacts produced by students after differential training against expert models in order to quantify the effects of the training. BODY.METHODS: BODY.CURRICULAR DEVELOPMENT AND IMPLEMENTATION: The curriculum was designed following a six-step approach, which has been designed for medical education [22], [36] but is not limited to this domain. These steps are general needs assessment, needs assessment of targeted learners, goals and measurable objectives, educational strategies, implementation, and evaluation and feedback. The definition of educational objectives is crucial for every educational process. Educational objectives essentially depend, among other factors, on the general requirements, the specific requirements of targeted learners and subject matter. Regarding the former two requirements, we outlined the general need to improve the ontology development quality and we have elucidated which components are necessary to achieve this goal. The needs of targeted learners who are domain experts and students from the life sciences depend heavily on their knowledge of computer sciences and formal logics, as well as on the subject matter they intend to represent. Since we did not want to overburden the curriculum, we restricted the group of targeted learners to students in life sciences with a background in computer sciences for which we could take basic knowledge and skills for granted. The subject matter addressed in the learning objectives had been operationally defined by the GoodOD guideline which comprises the basics of philosophical and formal ontology, descriptions logics, minimal metadata recommendations, top level ontologies and ontology design patterns, intended as a basic toolkit for ontology developers in any domain. Furthermore, learning objectives were formulated addressing the prevention of ontology development errors described in the introductory section of this paper. As an activity, ontology development requires the learners to acquire and shape knowledge of the domain to be represented, master the representation formalisms, as well as the skills of how to actually perform the task of creating a formal model. Therefore, we chose an instructional format which actively engaged the learners in a sequence of brief hands-on exercises, to be done individually or in small groups. Classroom lectures were minimized to short introductory talks. Every step of the curriculum was accompanied by printed handouts. Each instructional unit had duration of 105 minutes, each day consisting of four units. The complete seminar was designed for five and a half instruction days, corresponding to 22 units. Figure 1 shows the module sequence of the curriculum. 10.1371/journal.pone.0061425.g001Figure 1BODY.THE SEQUENCE OF MODULES IN THE CURRICULUM.: It follows the stepwise layout of the GoodOD guideline and the increasing complexity of the contents. Modules 10–13 and 15–16 were used in the intervention (see Table 1). BODY.STUDY DESIGN, POWER ANALYSIS AND ALLOCATION: An educational randomized controlled study was conducted in association with the curriculum as described above in September, 2011, at the Institute of Medical Biometry and Medical Informatics of the University of Freiburg, Germany. The complete curriculum included the interventional part (see below) and was completed in 5.5 days. Study design and reporting follows the CONSORT statement for randomized controlled trials in parallel group design (see Figure 2) [37]. 10.1371/journal.pone.0061425.g002Figure 2BODY.MODIFIED CONSORT DIAGRAM.: Twelve students were allocated to each group and could be analyzed. Prior to recruitment, power analysis for trials in parallel group design was performed using the R statistical package. The estimation of standardized effect sizes (Cohens’ d = mean differences/pooled standard deviation) for similarity measures was difficult because no empirical data sets were available which allowed a calculation of the mean differences and standard deviations to be expected. A sample size of n = 12 participants per group was calculated for parallel group design under the estimation of a high effect size of d = 1.2, a power of 0.8 and a significance level of 0.05. 24 students from four European countries (Austria, Germany, Slovenia and Switzerland) were included in the study. The students were recruited from universities which offer bachelor or master degrees in biology in combination with a minor subject in computer sciences or bachelor or master degree in computer sciences in combination with a minor subject in the life sciences. In every case inclusion criteria for participation were checked: applicants combined undergraduate knowledge in the life sciences with basic knowledge in computer sciences. Balanced randomization was performed with a pseudo random number generator from the R statistical package prior to the first interventional training sessions (see Figure 2). BODY.INTERVENTION: The intervention proper was conducted after the phase of background teaching in which the introductory modules of the curriculum were taught jointly to both groups. The intervention consisted of the differential training students received for certain topics in ontology design. For the modules 10–13 and 15–16 the students were either instructed or not. The allocation of modules to each group is displayed in Table 1. Modules 10–13 are dedicated to the proper usage of top-level categories (taken from BioTop) and corresponded directly to sections in the guidelines. Group A was trained on “Process and Participation” (module 10) and on “Immaterial object” (module 12), whereas group B was trained on “Collective material entity” (module 11) and “Information object” (module 13). Modules 15 and 16 covered the application of ontology design patterns; they also corresponded directly to sections in the guideline. Here, group A received training on the Closure ODP (module 15) and group B on the Spatial disjointness ODP (module 16). 10.1371/journal.pone.0061425.t001Table 1BODY.INTERVENTION AND DATA COLLECTION OF THE STUDY.: Intervention/data collection Application of top-level ontology Application of ODPs Training group A Module 10 (PRO) Process and participation Module 12 (IMM) Immaterial object Module 15 (CLO) Closure ODP Training group B Module 11 (CME) Collectivematerial entity Module 13 (INF) Information object Module 16 (SPA) Spatial disjointness ODP Test exercises both groups PRO: Photosynthesis, Medical diagnosing; CME: Proteinuria,Penicillin IMM: Fetogenesis, Stomach anatomy; INF: Operation plan, Pneumonia diagnosis SPA: Cell membranes, Stomach wall; CLO: Circulatory system, Teeth The intervention of the study consisted of the differential training of the students in certain content areas: students in group A received training in modules 10, 12 and 15 and no training in modules 11, 13 and 16, and vice versa for students in group B. Training sessions were kept balanced with regard to instructor, length, difficulty and instructional format. Data were collected in the form of ontology development exercises which were distributed evenly over all content areas with two exercises per training module. Training sessions were kept balanced with regard to instructor, length, difficulty and instructional format. The training sessions were held in parallel for both groups by two instructors for Modules 10–13. Instructors switched groups between these interventional training sessions to minimize trainer bias. Modules 15 and 16, the two training sessions on ontology design patterns, were given by the same instructor. Basically, sessions were as closely balanced as possible between the two groups for training duration, complexity and instructional structure. The study design introduced above has a crossed interventional schema: Groups received both the same interventions (guideline-based training or no-training) but on modules with different topics, respectively. The crossed design has been chosen in this educational study to control context dependency of the effect which is introduced by the topic of the module. It is possible that an effect depends only or partly on the topic of an educational intervention and not on the intervention proper (here: training/no-training). To control this effect, more than one module (topic) was selected in each group and the design was crossed so that each group received training on different modules respectively. For practical and ethical reasons, a pure parallel group design was impossible, however theoretical possible, in which one group would have received training on all modules and the other group no training at all. In this study, it was not intended to compare the overall effect of training between different modules. Only the effects of guideline-based training vs. no-training were compared between the groups on same modules. However, this design has some limitations discussed below. BODY.OUTCOME MEASURES AND INSTRUMENTS: Primary outcome measure was the mean similarity of ontology artefacts developed by the students in the assessment exercises compared with a set of gold standard artefacts provided by the authors. Prior to the experiment the authors had prepared two test exercises for each of the six interventional training topics. All exercises were given in the same format and provided with the following material: (a) an introductory text explaining the exercise; (b) an OWL file containing the necessary ontology primitives; and (c) the upper level ontology BioTopLite, a simplified version of the upper level ontology BioTop (to be imported by (b)), which corresponded to the students’ knowledge from the training sessions (individualized for each group). The learners had to arrange the ontology classes in a taxonomic order and add describing and defining axioms according to the given task. The creation of new object properties (relations) was not allowed. The contents of the tasks and their allocation to the training sessions are presented in Table 1. In order to measure ontology similarities different instruments were combined [39]. Prior to calculation of metrics, a modified normalization approach was applied to minimize syntactic differences between ontologies, which did not affect their intended semantics [40]. Precision, recall and f-measure were determined according to Dellschaft and Staab [34], for which we have developed a software library [39]. Another library, OntoSim, was used to calculate triple-based entity similarity in combination with average linkage or minimum weight maximum graph matching (MWMGM) similarity [35], [41], [42]. Secondary outcome measures were the intra-group ontology similarity metrics between artefacts created by students who had either received the training or not (intra group homogeneity between ontologies). Measuring instruments were the same as for the primary outcome. BODY.DATA COLLECTION: Data were collected during assessment sessions held in two and a half days directly following the training sessions in the structure as presented in Table 1, in which the participants processed the exercises. After each session the individual ontology artefact produced by each student was collected as an OWL file and stored in its original state so that each assessment session yielded 24 ontology artefacts. All 12 result OWL files from each of the 24 participating students were collected, totalling 288 files. BODY.STATISTICAL ANALYSIS: A Java program was written to pre-process the files and derive metrics as described above [39]. For each measure and each exercise a tabular output file was produced so that each file contained 24 = 576 similarity/distance values corresponding to the mutual pairs of each of the 24 student results and the respective gold standard ontology. For further aggregation, statistical procedures, and the production of graphs the R statistical package (version 2.15.1) was used [38]. The individual distance measures were aggregated by calculating means and standard deviations for each student inside the own group, so that aggregated measures for the distance of the ontologies in the own group resulted. Based on the individual measures for the distances to the gold standard and the calculated distances to the collection of ontologies in the own group, further parallel group analysis of aggregated means on the level of single ontologies were performed. To estimate differences between treatment groups at the level of each ontology artefact, the treatment effect was calculated as the difference between the group means of distances to the gold standard and between the intra-group mean distances. The statistical significance was tested using the t-test for independent samples. BODY.ETHICAL APPROVAL: Participants received an expense allowance of 500 for their participation in the training and the study. Before their agreement, students had been informed about all details of the curriculum and the following study. As part of the agreement, it was explicitly stated that the payment of the allowance was only dependent on the students’ complete attendance and full cooperation during the training sessions and the study but not on their success in the assessments or answers in the questionnaires. Ethical approval was requested from the ethical authority of the University of Freiburg, Freiburg, Germany. The chair of the University of Freiburg ethics committee reviewed the project and concluded that a full formal ethics committee statement was not required due to the educational nature of the study. It was designed according to the general requirements for educational studies at the University Medical Center Freiburg, Freiburg, Germany, and was performed with written informed consent of the participants. BODY.RESULTS: To our knowledge there are no prior studies which obtained similarity measures between ontologies developed by study participants and gold standard ontologies provided by experts after specific training on ontology design as metrics for the improvement of ontology development skills. In this randomized controlled study we sampled ontology artefacts from 24 students addressing 12 modelling task for which they had been either trained for or not prior to the assessment. We calculated similarity measures of these sample ontologies with gold standard ontology artefacts and with the artefacts of other participants inside the same training group (intra-group homogeneity). The obtained data did not provide support for our main hypothesis, viz. that a guideline based ontology training improves the developers’ skills to build ontology artefacts that were more similar to pre-existing gold standards. The artefacts from the trained group were not significantly more similar to the gold standards as the ones from the untrained group. As clearly shown in Table 2, Table 4 and Table 5, we could find only very small and not statistically significant treatment effects between the trained group and the untrained group. This result is consistent for all ontology similarity metrics and ontology assessment tasks (Table 2). Even with aggregated data on topic levels only small and not significant effect sizes were observed (Table 5). 10.1371/journal.pone.0061425.t002Table 2BODY.SIMILARITY WITH THE GOLD STANDARD MODEL.: group topic ontology fm n-mtb mtb atb A CLO tee −0.4 −3.8 −0.1 −0.3 A CLO bud 6.6 0.6 −2.5 6.9 A IMM sto 11.1 1.0 −1.5 −0.1 A IMM fet −5.4 2.0 0.3 1.2 A PRO pho 1.6 1.6 −0.4 −2.8 A PRO dia 1.7 2.2 −1.0 2.4 B CME pru −4.7 0.1 0.0 0.0 B CME pen −3.6 −0.9 1.4 0.1 B INF pne −6.0 2.0 −0.1 −1.1 B INF ope 1.0 1.5 −0.1 0.0 B SPA cem 0.5 −1.4 0.0 1.4 B SPA sta 7.0 4.1 −0.8 1.5 Ontology similarity metrics, displayed as absolute difference in percent between trained and untrained groups ordered by trained group, training topic and individual assessment task (for details see Table 1). The similarity/distance metrics shown are f-measure (fm), MWMGMS after normalization (n-mtb), MWMGMS without normalization (mtb) and average linkage without normalization (atb), the last three combined with triple-bases entity similarity as local measure. Significance levels of group comparisons are indicated as ∼: p0.15, : p0.1, : p0.01, : p.001. 10.1371/journal.pone.0061425.t003Table 3BODY.EFFECT-SIZES OF SIMILARITY WITH THE GOLD STANDARD ONTOLOGY AND INTRA-GROUP SIMILARITY.: group topic ontology GS-fm[%] GS-fm d IH-fm[%] IH-fm d A PRO pho 1.6 0.12 9.7 0.66 A PRO dia 1.7 0.12 6.5 0.35 A IMM sto 11.1 0.63 11.3 0.59 A IMM fet −5.4 −0.30 2.3 0.16 A CLO tee −0.4 −0.03 −3.0 −0.20 A CLO bud 6.6 0.51 4.1 0.31 B CME pru −4.7 −0.38 8.0 −0.51 B CME pen −3.6 −0.34 −5.2 −0.43 B INF pne −6.0 −0.46 −2.3 −0.23 B INF ope 1.0 0.05 −13.0 −0.69 B SPA cem 0.5 0.04 −9.0 −0.80 B SPA sta 7.0 0.37 −0.2 −0.01 F-measure ontology similarity metrics with the gold standard (GS) and f-measure intra-group homogeneity (IH), displayed as absolute differences in percent between trained and untrained groups and Cohens’ d effect sizes. Ordering is by trained group, training topic and individual assessment task. For details on abbreviations and symbols see Table 2. 10.1371/journal.pone.0061425.t004Table 4BODY.AGGREGATION OF EFFECT-SIZES ON TOPIC LEVEL.: group topic GS-fm [%] GS-fm d IH-fm [%] IH-fm d A PRO 1.6 0.12 8.1 0.49 A IMM 2.8 0.16 6.8 0.40 A CLO 3.1 0.23 0.5 0.04 B CME −4.1 −0.36 −6.6 −0.47 B INF −2.5 −0.16 −7.7 −0.50 B SPA 3.7 0.24 −4.6 −0.23 F-measure ontology similarity metrics with the gold standard (GS) and f-measure intra-group homogeneity (IH) aggregated on topic level, displayed as absolute differences in percent between trained and untrained groups and Cohens’ d effect sizes. Ordering is by trained group and training topic. For details on abbreviations and symbols see Table 2. 10.1371/journal.pone.0061425.t005Table 5BODY.INTRA-GROUP SIMILARITY (HOMOGENEITY).: group topic ontology fm n-mtb mtb atb A PRO pho 9.7 0.1 −0.5 −0.3 A PRO dia 6.5 0.1 −0.8 −0.9 A IMM sto 11.3 −0.3 0.0 −1.3 A IMM fet 2.3 −1.2 0.0 0.4 A CLO tee −3.0 −0.7 0.0 −0.4 A CLO bud 4.1 0.4 1.0 −1.0 B CME pru −8.0 0.1 0.0 0.0 B CME pen −5.2 −1.4 0.3 1.0 B INF pne −2.3 −0.2 0.0 0.1 B INF ope −13.0 −0.7 0.1 0.3 B SPA cem −9.0 0.3 0.0 −0.2 B SPA sta −0.2 1.1 −0.7 −0.2 Intra-group ontology similarity metrics (homogeneity), displayed as absolute difference in percent between trained and untrained groups ordered by trained group, training topic and individual assessment task. For details on abbreviations and symbols see Table 2. Our second hypothesis that the homogeneity between ontologies from different developers would increase due to guideline based training when compared with the untrained group was not supported by our data either (Table 3, Table 4 and Table 5). Although we observed moderately significant effect sizes for the f-measure intra-group similarity, the overall comparison between the groups is not conclusive. For group A the f-measure increases significantly about 5% (absolute) after training but for group B it decreases significantly about 6% (Table 5). For the other tested similarity measures only small differences between the trained and untrained group of lower than 1.5% were found. Why the f-measure metrics are more sensitive for intra-group similarity differences than the other metrics remains unclear and should be subject of further investigation. Contrary to our prior assumptions, we did observe differences in intra-group similarity between trained and untrained students in both directions. However, the direction of the differences might depend on the topic of the training session and corresponding assessment tasks or the (random) allocation of students to the groups. The exact cause is however not determinable with our study design. At this point, we are not able to confirm our assumption that skills of novice ontology developers are improved by guideline-based training. Moreover, our results indicate that the content topic of the training and the corresponding assessment task are factors that might dominate the training effects. Furthermore, despite our best efforts those content topics may have been distributed in an imbalanced way in the two groups and may have caused the different effects in the intra-group f-measure similarity metrics. BODY.DISCUSSION: Our research question for this randomized controlled trial was whether the skills of ontology developers can be improved by a guideline-based training when compared with developers who received unspecific training. To provide empirical evidence to support this hypothesis, we have developed a curriculum based on a guideline on good ontology design and evaluated its effect in a randomized controlled trial. The primary outcome parameter of the study was the similarity of ontologies developed by participants with a gold standard model provided by the authors of the guideline. The secondary outcome parameter was the intra-group homogeneity of ontologies. The data of our study did not provide evidence for either hypothesis; neither did the similarity to a gold standard model increase significantly nor did the intra-group homogeneity of participants change consistently. There are several possible causes for the difficulty to detect an unambiguous effect of our training on the quality of ontologies as the product of the development process. On the one hand, the explanatory power of the study was limited due to the study design. However, this argument does not account for the failure to even detect a difference in similarity to the gold standard model. On the other hand the deployed similarity metrics might be too insensitive to detect slight differences in features present in our ontologies. The simplest explanation for the result of our study would be that students were overloaded with too much information in too short a time. The complete interventional training sessions were given in 2.5 days. Although they consisted of two 105 min units for each instructional topic and an additional free training session, they were overly ‘packed’ with information. Thus, students possibly had not enough time to consolidate their recently acquired knowledge in a longer training session. Moreover, the recall of fresh knowledge and skills might even be inhibited by the task to tackle new problems after only a short interval to a prior learning experience that was not yet fully consolidated (retroactive interference) [43]. However, the results of our study should not be considered to indicate a complete failure of the guideline-based approach of ontology development. As an evaluation of a guideline, our study had objectives on two levels. First, it had the objective to provide evidence on the efficacy of a guideline-based training on the performance of ontology developers. On top of that, a secondary objective was to show that the guideline-based approach is superior to the ‘conventional’ more or less unguided development of ontologies. What we can state here, is that our training was not effective, but that does not imply that the guideline-based approach, in general, is not effective. As we outlined above, our interpretation is that the educational intervention and the measurement instrument were insufficient to show small differences in a complex environment. BODY.LIMITATIONS OF THE STUDY: As an educational study in a complex environment, this study has some limitations which render the interpretation of its results very difficult. From a pure educational standpoint, a comparison of an educational intervention with an unspecific instruction in the control group was criticized to be low-stakes educational research [44]. Arguing from this standpoint alone, a positive result would have meant only that the provided educational intervention ‘had been better than nothing’ – which is really not much. However, as a study in guideline evaluation we designed it to show the effectiveness of the guideline-based approach. Seen from this more complex standpoint, a positive result could have been interpreted as: (1) the provided guideline-based intervention ‘had been better than nothing’, and (2) the guideline-based approach to ontology engineering is better than a non-guided approach. We faced the problem to show a clear effect under the condition of a small sample size and a complex educational and technical setting. However, a larger sample size was not feasible due to practical limitations. It was already difficult to recruit the participants of the present study, and a larger group size would also have impeded the educational implementation. There would have been two alternative study designs with higher statistical power: Within the parallel group design we could have chosen to assign all interventions to the same group. Most probably this would have decreased the spread of our results and thus increased the statistical power of the result. However this would have had two important disadvantages: It would have been a hindrance for recruitment, as half of the participants would have learned considerably less during the summer school (and learning is one of the key incentives for participation in such an event). Moreover, this line would have increased the ‘better than nothing’ problem of educational studies. Statistical significance would have also been higher, if we had evaluated our data by using the crossover method [45], [46]. Although the conditions were kept as closely similar as possible for the two groups, a major structural limitation of an educational crossover design lies in the training and assessment of different content areas. When sequential crossover studies are performed in pharmaceutical studies, a washout period can be inserted that is long enough to reach complete extinction of the treatment effect of the first treatment period. In educational crossover studies, however, a back-learning (forgetting) is neither possible nor desirable, so that the instructional methods that are to be compared must be applied to different content areas. Although we switched the roles of intervention group and control group for different treatments, we decided not to analyse the results as a crossover study because we considered the topical differences of the educational interventions as too large. Even this way, and however carefully chosen for similarity in difficulty, length and amount, the content itself introduces a confounding variable in our design in which training for different content was ‘switched’ between groups. Although we tried to match the training topics of ontology design to have comparable content distributions for each group, they remain markedly different due to the character of the topics (Table 1). We consider this heterogeneity in content a possible reason for the different results on intra-group homogeneity in the two groups. As further elaborated in the next section, no validated and tested evaluation methods for computerized measurement of quality indicators of a larger number of ontologies are available. The resources of this project limited us to employ existing methods without prior prototyping of the study. Thus we missed the possibility to detect the limitations of the ontology similarity/distance metrics which were originally introduced for the evaluation of ontology mapping experiments. BODY.EVALUATION OF AND METRICS FOR FORMAL ONTOLOGY: Due to the complex nature of the ontology artefacts, their evaluation is inherently difficult and manifold. Furthermore, there are often multiple correct ways to represent the same facts, and the objectives and methods of ontology evaluation are numerous [32], [47], [48]. What do we exactly mean when we talk about ‘good ontology’? This property might be dependent on the objectives of the ontology under scrutiny, its philosophical foundations and the intention of the investigator. It is possible for two serious investigators to rate the same ontology on opposite poles of a quality scale [49]. In our view, the quality criteria for ontologies should be defined in accordance with their real or hypothetic application scenarios, besides the mere formal correctness of an ontology, which is already a starting point for good quality. However, as our interest lies in biomedical ontology, we can define several parameters for good quality ontology which are independent of their primary intended application scenario: Consideration of a continuous and appropriate ontological commitment throughout the ontology development process. The integration of a top-level ontology [21], [50]–[52] in the domain ontology can guarantee for this adherence in a more robust way as the usage of certain ontology design patterns, as the top-level ontology already provides many patterns in an embedded way, ensuring all patterns harmonize with each other, i.e. fit a common scope and development philosophy. Adherence to coding standards and naming conventions as defined by policy and best practice providers [8], [53]. Correct and exhaustive representation of the domain that is intended to be represented. Although a variety of metrics and methods could be useful in ontology evaluation, it is not clear how to measure certain properties, which might be used as quality indicators as listed above. In the case of our experiment, we can judge the quality of the produced ontologies by observing selected features such as the structure of the taxonomy, the axiomatization, representational errors, completeness, and correctness. However, to quantitatively assess the structure of the taxonomy or axiomatization in an objective and automatically manner seems to be impossible with the given tools. Our measurement hypothesis to compare the learners’ ontology artefacts with a gold standard, and to apply ontology similarity metrics, seemed most adequate to capture the two latter points. Unfortunately, we cannot claim that this strategy was successful. In the end, these methods, which were originally developed for ontology alignment experiments, were too insensitive to detect relevant differences between the test and gold standard ontologies. BODY.FURTHER RESEARCH: Future research should develop ontology evaluation methods that are able to quantify quality parameters of ontologies in accordance with a specific description logic expressivity regime (affected by the application use case and reasoning style). These quality parameters must be empirically validated for their explicit scopes. One direction of ontology evaluation can be the deployment of competency questions [54] which is a way to formalize requirements and assess their fulfilment. An automatic and objective evaluation of ontology may be possible with a strong formalization of competency questions as logical expressions. Further research should also yield empirical evidence for the efficacy of certain beliefs, dogmas and practices in ontology development. Ontology development is not an ‘artistic craft’ but an engineering activity which should be led by a high level of evidence and not only by expert opinion in analogy to practice in evidence based medicine [55]. How should quality indicators for ontologies be operationally defined? The overall quality of an ontology is dependent on a variety of factors. Hence, quality indicators should at least be defined to provide useful measures for ontology development and deployment; furthermore they should be unambiguous and measurable in a reproducible way, ideally automatically to avoid any bias. How can the quality of ontologies be measured computationally? Although many methods for ontology evaluation have been described or proposed, many open questions remain on how to measure quality indicators of ontologies technically, reproducibly and automatically. To support good quality ontology design, there should be an easy-to-use toolbox of ontology metrics which can be applied from the ontology editor of choice. Using such a toolbox, the developers can evaluate their ontology artefacts at each step of the development process and judge on how to proceed. How effective are specific ontology designs? It is not enough to claim that a certain type of development, top-level ontology, taxonomy or ontological commitment will result in better ontologies. At present, such claims are often brought forward without any empirical evidence. To overcome this situation, it is desirable to provide empirical evidence which methods of ontology design are more effective to reach specific objectives. How can the behaviour of ontology developers be changed effectively? Evidence on good practice is not enough. Ultimately, good practices and new methods of development have to be implemented in the ‘community of developers’. To understand and guide those complex socio-technical processes it must be understood how to change thinking, behaviour, communication and tools of developers. To investigate this complex network more educational studies are necessary. Furthermore, this educational research must adhere to the premise of proceeding incrementally by strictly changing only one parameter at a time in the experimental setting of each study [56]. BODY.CONCLUSIONS: Inadequate knowledge and insufficient skills of ontology developers are among the causes for quality problems of ontologies in the biomedical domain. To improve this situation, we developed GoodOD, a guideline for good ontology design, optimized for the development of OWL ontologies using description logics for representation and reasoning. We implemented a training course based on this guideline. In a randomized controlled trial with 24 students we investigated the efficacy of the guideline based training on the quality of developed ontology modules and patterns. We could not detect enhanced quality in terms of similarity to gold standard representations of the ontology artefacts produced after the selective training, when compared to artefacts built without prior training. Due to the study design outlined in this paper, the interpretation of such study results is difficult. Although we could not provide evidence for the effectiveness of a guideline-based approach to ontology design, the study neither provides evidence against the efficiency of this approach. More effort has to be invested in further research which can reliably provide empirical evidence whether specific development guidelines and practices in ontology design are effective.

TITLE: A Comparative Study on the Effects of Vintage Nonpharmacological Techniques in Reducing Myopia (Bates eye exercise therapy vs. Trataka Yoga Kriya) ABSTRACT.BACKGROUND:: Human eye captures light rays as they come and fall on the retina and convert them into an image. However, in myopia, light rays fall in front of retina, causing blurring of image. Correction of this is generally done using correcting devices such as corrective glasses and contact lenses. Existence of some alternative therapies is also noticed in literature. ABSTRACT.AIM:: To compare the effects of Bates eye exercises and Trataka Yoga Kriya on myopia. ABSTRACT.MATERIALS AND METHODOLOGY:: Ethical clearance was obtained from the institution, and informed consent was taken from participants. In this randomized comparative study, 24 participants (48 eyes) were taken based on inclusion and exclusion criteria and were randomly divided into two groups: Group A and Group B, where Bates eye exercise therapy and Trataka Yoga Kriya were given, respectively, for 8 weeks. Participants were assessed for their refractive errors and visual acuity pre- and post-intervention. ABSTRACT.RESULTS:: Data were analyzed by SPSS version 20. Results obtained revealed that both Bates exercises and Trataka Yoga Kriya were not significantly effective in reducing refractive errors and in improving visual acuity (P value of refractive error in right eye: 0.4250; left eye: 0.4596; P value of visual acuity in right eye: 0.5691; left eye: 0.8952). ABSTRACT.CONCLUSION:: This study concludes that nonpharmacological approaches such as eye exercises and Trataka Yoga Kriya are not significant on myopia. BODY.INTRODUCTION: In myopia that is also called as short sightedness, there is a problem in the refraction of light, in which when the accommodation is at rest, the parallel rays of light from the space come to focus in front of retina.[1] For the visual disability throughout the world, it is the common cause.[2] About 69% of the general population in India has been reported to be affected with myopia.[3] The prevailing treatment of refractive errors which are commonly used these days are glasses, contact lenses and refractive surgeries. The thought of substitution of the prevailing modalities of treatments leads the thinkers to the nonpharmacological therapies such as “Bates eye exercise therapy” and some ancient techniques such as “Trataka Yoga Kriya.” Some ocular strengthening therapies are involved in Bates method, by which the ocular health can be maintained and promoted. However, this kind of therapy is yet underevaluated by the modern sciences.[2] The mainstream ophthalmologists rejected the Bates method during his generation and remains as same even today.[45] Anyhow, it is seen that the eye exercises strengthen the eye muscles, and thus, diplopia and the point of convergence decrease. Meanwhile, Trataka Yoga Kriya of the ancient Indian tradition has been practiced throughout India and is strongly believed to increase the eye sight.[6] In the present research scenario, due to the lack of evidential studies over nonpharmacological treatment approaches in treating myopia, there is a need to study for different alternative approaches. This will help in extending the scope of physical therapy in modern medicine. Hence, the purpose of this study is to compare Bates exercises and Yoga in patients with myopia. BODY.OBJECTIVE: To evaluate the effects of Bates eye exercises and Trataka Yoga Kriya on myopia by comparing the changes in refractive errors and visual acuity. BODY.MATERIALS AND METHODS: A comparative study was conducted for 6 months on a sample size of 48 participants with myopia that was calculated using the G-power software. The inclusion criteria were an age group of 18–25 years, a refractive index ranging from −1.00 to −10.00 diopters, both genders, i.e., male and female, and myopia for the past 5 years, and the exclusion criteria were any lenticular or corneal opacity, other known ocular pathology, and infantile myopia. After receiving informed consent, participants were assessed by an ophthalmologist and the baseline values of outcomes were noted. The assessor was blinded and through simple randomization divided the participants into two groups with 24 participants and/or 48 eyes each. Participants were taken for a subjective and objective assessment before and after the treatment. The materials used in this study were Snellen's chart, lenses tray, autorefraction equipment, room temperature water, ice cold water, napkins, and candles. Intervention was given for 8 weeks; at the end of which, participants were assessed by the same ophthalmologist. BODY.PROCEDURE: In the Group A, the participants performed eye exercises for 8 weeks. The exercises given to this group were based on Bates technique; they were near to far shifting, palming, solarization, eye wash, eye squeezing, eye circles, and eye massage. In the Group B, Trataka Yoga Kriya was performed by the participants for 8 weeks. The following activity was done by the participants of this group for 8 weeks. Trataka means steady gazing. It can be done on any external objects such as a candle, a leaf, a crystal, and a Shiva Linga. This included two steps; they were Antaranga Trataka and Bahiranga Trataka. BODY.RESULTS: The results of this study were analyzed using the SPSS software version 20, in which the means and standard deviation of the normally distributed data of the participants were calculated. The graphs with P values and t-values were plotted using the GraphPad InStat 3.10 by GraphPad Software. There was no statistical significance in the postintervention P values of the data obtained for both the groups receiving Bates eye exercises and Trataka Yoga Kriya, in terms of refractive errors and visual equity of the participants with myopia. The results of the study are interpreted in the form of tables and graphs as follows. BODY.INTRAGROUP COMPARISON OF REFRACTORY ERRORS AND VISUAL ACUITY: The Table 1 and 2 depicts the intra group comparison of refractive errors and visual acuity within the groups respectively. Table 1Refractive errors within the groups Table 2Visual acuity within the groups BODY.INTERGROUP COMPARISON OF REFRACTIVE ERRORS AND VISUAL ACUITY: The two-tailed t-test has been incorporated to assess the p-values of post-intervention of refractive errors and visual acuity among both right and left eyes respectively. Figure 1 depicts the P value (0.425) of postintervention for refractive errors in the right eyes for the Group A and Group B. Whereas, Figure 2 depicts the P value (0.459) of postintervention for refractive errors in the left eyes for the Group A and Group B respectively. Figure 1P value of postintervention in the right eyes of Group A and Group B (refractive errors) Figure 2P value of postintervention in the left eyes of Group A and Group B (refractive errors) Figure 3 depicts the P value (0.656) of postintervention for visual acuity in the left eyes for the Group A and Group B. Whereas, Figure 4 depicts the P value (0.982) of postintervention for visual acuity in the right eyes for the Group A and Group B respectively. Although none of these P values are statistically significant. Figure 3P value of postintervention in the left eyes of Group A and Group B (visual acuity) Figure 4P value of postintervention in the right eyes of Group A and Group B (visual acuity) BODY.DISCUSSION: Myopia is a leading cause of loss of vision throughout the world, and its prevalence is increasing. The literature review shows that previous studies have stated that there is great effect of different eye exercises on refractive error.[67] The reason behind the effect these eye exercises show is thought to be as it strengthens accommodation, which is the power of changing focus of eyes for vision. However, the basic concept behind Trataka Yoga Kriya is relaxation of mind and eye that in turn improves vision. In this study, it was seen that there was a slight reduction in the myopia by performing the eye exercises. Hence, it states that Bates eye exercise has significant effect over the refractive errors over the participants involved in this study. However, due to lack of evidential studies over this, Bates method and Trataka Yoga are not yet proved to have a significant effect in reducing myopia. The participants in this study got a very minimal reduction of power. Therefore, it could be considered because of stress relief phenomena. Eventually, selection bias (the participants with range of refractive error were wide) and subjective assessment errors might also be some of the reasons. Certain variables were uncontrolled in this study, and their effect influenced the results was assumed to be null. Whether or not the participants in the eye exercises group were actually doing the eye exercises in proper repetition and accuracy and the subjects in the Trataka group, how far were able to imagine the image of the candle flame in their inner vision. The lifestyle and dietary habits of the participants were assumed to have no effect on the muscle strengthening and improvement in the accommodation. The future directions would be that the results in this study could be applied over the population diagnosed with myopia. This study did not include long-term intervention and follow-up period though exercises are effective for long-term benefits of interventions. There is a need for more research to be done to provide strong evidence whether Bates eye exercises and Trataka Yoga are effective for the reduction of myopia. By observing the obtained results, the limitations would be lack of a larger sample size, short interventional protocol duration, the Trataka being tiresome for the participants to perform as they had to constantly gaze on the flame of the candle which could cause inconvenience for them; as the participants were students, they were asked to perform the exercises and the Trataka before and after the college hours, respectively. This caused inconvenience for the students to do the activities regularly. BODY.CONCLUSION: This study concluded that both Bates eye exercises therapy and Trataka Yoga Kriya in reducing refractive errors in myopia were not effective. There was no significant improvement found postintervention in the refractive errors and visual acuity among both the groups. BODY.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Effect and Safety of Morphine Use in Acute Anterior ST‐Segment Elevation Myocardial InfarctionBonin et al ABSTRACT.BACKGROUND: Morphine is commonly used to treat chest pain during myocardial infarction, but its effect on cardiovascular outcome has never been directly evaluated. The aim of this study was to examine the effect and safety of morphine in patients with acute anterior ST‐segment elevation myocardial infarction followed up for 1 year. ABSTRACT.METHODS AND RESULTS: We used the database of the CIRCUS (Does Cyclosporine Improve Outcome in ST Elevation Myocardial Infarction Patients) trial, which included 969 patients with anterior ST‐segment elevation myocardial infarction, admitted for primary percutaneous coronary intervention. Two groups were defined according to use of morphine preceding coronary angiography. The composite primary outcome was the combined incidence of major adverse cardiovascular events, including cardiovascular death, heart failure, cardiogenic shock, myocardial infarction, unstable angina, and stroke during 1 year. A total of 554 (57.1%) patients received morphine at first medical contact. Both groups, with and without morphine treatment, were comparable with respect to demographic and periprocedural characteristics. There was no significant difference in major adverse cardiovascular events between patients who received morphine compared with those who did not (26.2% versus 22.0%, respectively; P=0.15). The all‐cause mortality was 5.3% in the morphine group versus 5.8% in the no‐morphine group (P=0.89). There was no difference between groups in infarct size as assessed by the creatine kinase peak after primary percutaneous coronary intervention (4023±118 versus 3903±149 IU/L; P=0.52). ABSTRACT.CONCLUSIONS: In anterior ST‐segment elevation myocardial infarction patients treated by primary percutaneous coronary intervention, morphine was used in half of patients during initial management and was not associated with a significant increase in major adverse cardiovascular events at 1 year. BODY.CLINICAL PERSPECTIVE: BODY.WHAT IS NEW?: This retrospective study evaluating outcomes associated with morphine use in an ST‐segment elevation myocardial infarction population provides additional data that use of morphine is not associated with adverse outcomes. BODY.WHAT ARE THE CLINICAL IMPLICATIONS?: Use of morphine to relieve chest pain is safe in patients with ST‐segment elevation myocardial infarction. BODY.INTRODUCTION: Morphine is currently used and recommended for the treatment of chest pain during myocardial infarction, but the level of evidence is low, attributed to the lack of supportive clinical studies.1, 2, 3, 4 Moreover, the American Heart Association has relegated morphine use in patients with non‐ST‐segment elevation myocardial infarction from a Class I to a Class IIa recommendation.3 This modification was driven by the results from the CRUSADE (Can Rapid Risk Stratification of Unstable Angina Patients Suppress Adverse Outcomes with Early Implementation of the American College of Cardiology/American Heart Association Guidelines) registry showing that, in patients with non‐ST‐segment elevation myocardial infarction, morphine use increased the risk of death and adverse outcome.5 Morphine has also been associated with suboptimal reperfusion success and a low myocardial salvage index after primary percutaneous coronary intervention (PPCI) in patients presenting with ST‐segment elevation myocardial infarction (STEMI).6 Nevertheless, in 2 independent cohorts from the FAST‐MI (French Registry of Acute ST‐elevation and non‐ST‐elevation Myocardial Infarction) registry, prehospital morphine use in STEMI patients did not increase in‐hospital complications or 1‐year mortality.7 Because of this controversy, additional investigations are necessary. The aim of this study was to explore the effect of morphine on clinical outcomes in a population of patients with anterior STEMI referred for PPCI. BODY.METHODS: The data, analytical methods, and study materials will not be made available to other researchers for purposes of reproducing the results or replicating the procedure. BODY.STUDY DESIGN: We used the database of the recently published CIRCUS (Does Cyclosporine Improve Outcome in ST Elevation Myocardial Infarction Patients) trial8, 9 for a post hoc analysis assessing the effect of morphine use in the initial management of patients with anterior STEMI undergoing PPCI. CIRCUS was an international, multicenter, double‐blind, placebo‐controlled trial conducted between April 2011 and February 2014. The CIRCUS trial showed no significant effect of cyclosporine on the clinical outcomes of patients with anterior STEMI.9 CIRCUS study was performed in accord with the principles of the Declaration of Helsinki and the European guidelines for Good Clinical Practice. Approval was obtained from the ethics committees in the relevant countries, and written consent for data use was obtained from all patients. BODY.STUDY POPULATION: We analyzed data from the intent‐to‐treat population of the CIRCUS trial. Eligible patients (male and female) were aged ≥18 years and presented within 12 hours of onset of symptoms evocative of acute coronary syndrome (ACS). They were required to have ST‐segment elevation ≥0.2 mV in 2 anterior contiguous leads and to be scheduled for PPCI. Written informed consent was obtained from all patients. Exclusion criteria included cardiogenic shock or loss of consciousness during the prehospital period, known hypersensitivity to cyclosporine, known kidney or liver failure, pregnancy or absence of contraception in women of childbearing age, and any disorder associated with immunological dysfunction within the previous 6 months. BODY.STUDY TREATMENT: CIRCUS patients were divided into 2 groups with and without intravenous morphine before PPCI. In a post‐hoc analysis setting, we hypothesized that morphine use would be superior to the absence of morphine use in terms of primary outcomes. BODY.STUDY OUTCOMES: The primary outcome was the rate of occurrence of major adverse cardiovascular event (MACE) including cardiovascular death, heart failure, cardiogenic shock, recurrent myocardial infarction, unstable angina, and stroke during 1 year of follow‐up. Cardiogenic shock was defined as systolic blood pressure <80 mm Hg for >30 minutes, unresponsive to fluid replacement, and associated with signs of peripheral and end‐organ hypoperfusion. Heart failure was defined as clinical symptoms requiring initiation or intensification of heart failure treatment with intravenous administration of diuretics. Unstable angina was defined as the presence of acute chest pain associated with ST depression, or new onset of negative T waves, and no elevation of troponins. Secondary outcomes included rate of occurrence of individual MACE, all‐cause death, adverse left ventricular remodeling during 1 year of follow‐up, and the creatine phosphokinase (CPK) peak blood concentration in the 24 hours post‐PPCI. MACE were adjudicated by an event validation committee composed of at least 3 cardiologists/physicians blinded to treatment. Total CPK blood concentration was measured in peripheral total blood at the time of PPCI and 4, 12, and 24 hours post‐PPCI. CPK measures were conducted locally, in each participating center. CPK peak concentration was defined as the highest value collected during the 24 hours post‐PPCI. It was used as an indirect evaluation of infarct size.10, 11 Adverse left ventricular remodeling was defined as an increase of 15% or more of end diastolic left ventricular volume indexed to body surface area. End diastolic volume was measured at discharge and at 1 year by blinded expert readers. The measures were performed on echocardiographic data, according to the Simpson biplane rule. BODY.SUBGROUP ANALYSIS: Cyclosporine is an inhibitor of cerebral12 and intestinal13 P‐glycoprotein. P‐glycoproteins are involved in morphine intestinal absorption14 and neurological circulation.15 This P‐glycoprotein inhibition could lead to an increased effect of morphine.14 To evaluate the effect of this pharmacological interaction on our results, we compared the 4 subgroups: morphine with cyclosporine; morphine with placebo; no morphine with cyclosporine; and no morphine with placebo on MACE occurrence and on infarct size. We also explored morphine with cyclosporine interaction effect on MACE occurrence by 2‐way ANOVA with an interaction term. BODY.STATISTICAL ANALYSIS: All statistical analyses were post hoc and were not prespecified in the original CIRCUS Statistical Analysis Plan. Continuous variables with normal distribution are expressed as means and SDs. Continuous variables with a non‐normal distribution are expressed as medians and interquartile ranges. Normality was tested with the Shapiro–Wilk test. Categorical variables are expressed as percentages. The study population was divided into 2 groups according to morphine use. Comparison of baseline characteristics or outcomes was performed using the chi‐squared test or Fischer's exact test, as appropriate, for categorical variables. The Student t For all comparisons, a value of P<0.05 was considered statistically significant. When appropriate, 95% confidence intervals were calculated. All statistical analyses were conducted using STATA software (version SE 14.2; (StataCorp LP, College Station, TX). BODY.RESULTS: The intent‐to‐treat CIRCUS population included 969 patients.8, 9 Two patients were not included in our analysis because of lack of information on morphine use. Morphine was used before PPCI in 554 (57.3%) patients. Baseline characteristics (Table 1) and periprocedural characteristics (Table 2) were well balanced between groups with and without morphine use. There was a nonsignificant trend toward younger age and a shorter total ischemic time in patients receiving morphine. There was also a trend toward more heart failure and cardiogenic shock in the morphine group: 13.1% versus 10.2% of patients admitted in Killip 2 or 3 and 1.4% versus 0% of patients admitted with cardiogenic in morphine and no‐morphine group, respectively (Table 2). Table 1Characteristics of the Patients at Baseline All Patients (n=967)Patients With Morphine (n=554)Patients Without Morphine (n=413) P Value (Wilcoxon or Fisher's Test)Age, y 60±13 59±13 61±13 0.07 Male sex, % 82 82 82 0.73 Body mass index, kg/m2 27±4 27±4 27±4 0.14 Current smoker, % 42 43 41 0.39 Hypertension, % 37 38 37 0.74 Diabetes mellitus, % 13 13 13 1.00 Dyslipidemia, % 38 40 36 0.18 Previous myocardial infarction, % 6 6 5 0.67 Previous PCI, % 7 7 6 0.61 LVEF, % 47±10 47±10 48±10 0.16 Cyclosporine use before PPCI, % 49 51 46 0.12 Values are expressed as means±SD. LVEF indicates left ventricular ejection fraction (measured by echocardiography); PCI, percutaneous coronary intervention; PPCI, primary percutaneous coronary intervention. Table 2Periprocedural Characteristics Patients With Morphine (n=554)Patients Without Morphine (n=413)Killip class at admission 505 353 Class 1 432 (85.5) 317 (90.0) Class 2 57 (11.3) 29 (8.2) Class 3 9 (1.8) 7 (2.0) Class 4 7 (1.4) 0 (0.0) Total ischemic time, mean±SD (h) 3.99±2.39 4.94±3.07 Rentrop Grade 2 or 3 34/554 (6.1) 31/413 (7.5) Angiographic thrombus burden ≥3 358/533 (67.2) 267/395 (67.9) Area at risk, mean±SD (%)a 36.8±8.4 35.6±8.6 Proximal localization 252/552 (45.6) 157/403 (38.9) Multivessel disease 203/554 (36.6) 154/413 (37.3) Thrombolysis rate 33/554 (5.9) 27/413 (6.5) Stenting 492/554 (88.8) 362/413 (87.6) No reflow 30/554 (5.4) 25/413 (6.1) Final TIMI 548 403 TIMI=0 9 (1.6) 4 (1.0) TIMI=1 2 (0.3) 8 (2.0) TIMI=2 29 (5.3) 29 (7.2) TIMI=3 508 (92.7) 362 (89.8) Treatment at discharge Double antiplatelet treatment 511/544 (93.9) 380/408 (93.1) Beta‐blockers 507/544 (93.2) 370/408 (90.7) Statins 522/544 (95.9) 389/408 (95.3) ACEi 482/544 (88.6) 348/408 (85.3) ARB 10/544 (1.8) 15/408 (3.7) Calcium‐channel blockers 12/544 (2.2) 13/408 (3.2) Diuretics 138/554 (25.4) 105/413 (25.4) Aldosterone antagonists 147/544 (27.0) 82/408 (20.1) Values are expressed as numbers (percentages), unless otherwise specified. ACEi indicates angiotensin‐converting enzyme inhibitors; ARB, angiotensin receptor blockers; TIMI, Thrombolysis in Myocardial Infarction. aUsing the APPROACH angiographic score. At 1 year, 236 (24.4%) patients had experienced at least 1 MACE. There was no significant difference in occurrence of MACE between groups: 145 (26.2%) and 91 (22.0%) patients in the groups with and without morphine, respectively (P=0.15; Table 3). Cumulative Kaplan–Meier estimates for the first occurrence of MACE (Figure 1) were not significantly different between groups (P=0.10). Table 3Clinical Outcome After 1‐Year Follow‐up EventMorphine (n=554)No Morphine (n=413) P (Fischer's Test)Any MACEa 145 (26.2) 91 (22.0) 0.15 Cardiovascular death 29 (5.2) 20 (4.8) 0.88 Heart failure 110 (19.9) 70 (16.9) 0.28 Cardiogenic shock 30 (5.4) 19 (4.6) 0.66 Recurrent myocardial infarction 21 (3.8) 7 (1.7) 0.08 Unstable angina 15 (2.7) 8 (1.9) 0.53 Stroke 10 (1.8) 9 (2.2) 0.82 Values are expressed as numbers (percentages). MACE indicates major adverse cardiovascular events. aA patient with more than 1 clinical event was counted as having 1 MACE. Figure 1Kaplan–Meier curves for major adverse cardiovascular events (MACE). In the Cox model, morphine use was not associated with the incidence of MACE (hazard ratio=1.25; 95% confidence interval [0.96; 1.62]; P=0.10), even after adjustment for age, ischemic time, infarct size (CPK peak), initial and final Thrombolysis in Myocardial Infarction flow, sex, smoking, hypertension, diabetes mellitus, previous myocardial infarction, and Killip class (hazard ratio=1.04; 95% confidence interval [0.75; 1.45]; P=0.82). Incidence of individual MACE during 1 year was not significantly different between groups (Table 3; Figure 2). Figure 2Occurrence of major adverse cardiovascular events (MACE) and individual events after 1‐year of follow‐up. There was no statistically significant difference on MACE occurrence (P=0.56) and on infarct size (P=0.61) between the 4 different treatment subgroups (Table 4). Interaction term, even after adjustment, was not significant and so for each clinical outcome individually (Table S1). Table 4Subgroups Analysis, Evaluation of Morphine—Cyclosporine Interactions Morphine+Cyclosporine (n=266)Morphine+Placebo (n=246)No Morphine+Cyclosporine (n=178)No Morphine+Placebo (n=205) P (ANOVA)MACEsa 70 (26.3) 75 (30.5) 44 (24.7) 47 (22.9) 0.56 CPK peakb 3945±2638 4054±2677 4061±3182 3742±2571 0.61 CPK indicates creatine phosphokinase; MACE, major adverse cardiovascular events. aValues are expressed as numbers (percent). bValues are expressed as mean±SD. There was no significant difference in rates of all‐cause death at 1 year between groups (32 [5.3%] and 22 [5.8%] deaths in the groups with and without morphine, respectively; P=0.89). The unadjusted Kaplan–Meier hazard curve for 1‐year all‐cause mortality is presented in Figure 3. No significant difference was observed between the groups with and without morphine (P=0.77). Figure 3Kaplan–Meier curves for 1‐year all‐cause mortality. CPK peak blood concentrations after PPCI were comparable in both groups (4023±118 and 3903±149 IU/L in the groups with and without morphine, respectively; P=0.52; Figure 4). Figure 4Creatine phosphokinase (CPK) blood concentration evolution during the first 24 hours following primary percutaneous coronary intervention (PPCI)—median, first and third quartiles, and first and ninth decile are presented for each measurement. Mixed‐model analysis for morphine effect on CPK kinetic curve: P=0.09. Rates of adverse left ventricular remodeling were also similar in both groups (205 [37.0%] and 134 [32.4%] patients with adverse remodeling in the groups with and without morphine, respectively; P=0.21). BODY.DISCUSSION: In a large cohort of patients with acute anterior STEMI, morphine was used in half of patients before PPCI and had no significant effect on the composite primary outcome including cardiovascular death, heart failure, cardiogenic shock, recurrent myocardial infarction, unstable angina, and stroke. Few other studies have evaluated the clinical outcomes of patients who received morphine to alleviate chest pain during an ACS.16 In line with our results, 2 retrospective studies concluded that morphine/intravenous narcotics did not adversely affect the outcomes in patients with ACS. In 1 study including 1758 patients (765 STEMI and 993 non‐ST‐segment elevation myocardial infarction), the rate of 30‐day death was not increased with the use of intravenous narcotics.17 In the second study, the analysis of 2 cohorts of STEMI patients (2438 patients from the FAST‐MI 2010 registry and 1726 from the FAST‐MI 2005)7 suggested that prehospital morphine use was not associated with increased in‐hospital complications and 1‐year mortality. On the contrary, an American retrospective study based on the CRUSADE registry,5 which included 57 039 patients who presented with non‐ST‐segment elevation ACS, reported an increase in mortality and challenged the safety of morphine use in these patients. It is difficult to compare the CRUSADE study with ours because of differences in population, study dates, and design. The CRUSADE study retrospectively enrolled non‐ST‐segment elevation ACS patients, who are different from STEMI patients. It was conducted between 2002 and 2003, at a time when oral antiplatelet agent use was limited to clopidogrel, which has more pharmacological interactions with morphine than more recently approved agents such as ticagrelor and prasugrel.18, 19 Thus, it seems that there is currently limited evidence for adverse outcomes associated with morphine use in STEMI patients, whereas additional caution may be necessary in NSTEMI patients. In our study, morphine use did not appear to be associated with any significant cardioprotective effect. Myocardial protection with opioid use has been inconsistently reported. Several studies suggest a cardioprotective effect especially with morphine in the surgical context of coronary artery bypass graft, but these studies were conducted in small surgical patient populations and assessed indirect outcomes.20, 21 In STEMI patients undergoing PPCI, a cardioprotective effect could be demonstrated for morphine in addition to the basal effect of remote ischemic conditioning,22 but no additional myocardial protection was observed with fentanyl in patients undergoing elective PPCI.23 In experimental models of ischemia reperfusion injury, morphine inhibits the mitochondrial permeability transition pore24 opening through μ and κ myocardial opioid receptors,25, 26 and induced a significant cardioprotective effect.27 Morphine also stimulates the reperfusion injury salvage kinase pathway.24 In animal studies, these mechanisms have been associated with a reduction of infarct size following intravenous or intrathecal morphine use before or immediately after reperfusion.28, 29, 30 However, the translation from animal models to humans is not straightforward owing to disease complexity and associated comorbidities, and our data do not suggest any cardioprotective effect of morphine on ischemia‐reperfusion injury. In the near future, 2 noncurrently published studies (Clinical Trials NCT01186445 and NCT01738100) may provide additional understanding of the cardioprotective effects of intracoronary morphine during ACS. In recent years, there has been a growing concern about the use of morphine for pain relief in patients with ACS. Morphine delays and attenuates the release peak and efficacy of oral antiplatelet agents in ACS31, 32, 33 and healthy18, 19, 34 patients, by inhibiting gastrointestinal absorption35 and inducing to vomiting.16 However, there is no evidence for the clinical relevance of these effects. In our study, although morphine was not associated with any adverse outcome in STEMI patients, there was a nonsignificant trend toward an increase in individual MACE in patients treated with morphine. In particular, recurrent myocardial infarction and heart failure seemed to be more frequent when morphine was used before PPCI, although these results did not reach statistical significance (P=0.08 and 0.28, respectively; Table 3). Regardless of the potential outcome modifications associated with morphine use during ACS, pain release is essential to decrease patients' discomfort and to avoid proarrhythmic anxiety. In our study, 42.7% patients did not receive any analgesia, meaning that pain was probably undertreated. This is not acceptable, and pain release should be a priority of any medical care. Without ethical possibility of a placebo‐controlled, randomized study, it is difficult to conclude definitively about morphine safety in this indication. There is no analgesic alternatives to morphine in this indication, and the only new approach currently evaluated is equimolar oxygen/nitrous oxide mixture (MEOPA) associated with paracetamol, under investigation in the ongoing SCADOLII (Comparison of MEOPA + Paracetamol Versus Morphine Treatment in Acute Coronary Syndrome Analgesia) trial. In the absence of clear evidence of adverse effects, morphine should be used without restraint in STEMI as was concluded in a recent review.16 BODY.STUDY LIMITATIONS: Although analyses were conducted on a prospective homogenous anterior STEMI population, they were post hoc analyses, not prespecified in the original CIRCUS protocol, leading to a loss of statistical relevance and limited statistical power. Given that CIRCUS trial randomization was not stratified on morphine use, the effect of unmeasured confounders on the results cannot be ruled out. Anyway, patients in the morphine group seemed to be sicker at initial medical care than those in the no‐morphine group: 13.1% of patients were admitted with heart failure (Killip 2 or 3) in the morphine group versus 10.2% in the no‐morphine group; 1.4% of patients were admitted with cardiogenic shock in the morphine group versus 0% in the no‐morphine group. There was no randomization, nor precise recommendation, for morphine use in the CIRCUS protocol. So, morphine could have been used as a symptomatic treatment, in combination with continuous positive airway pressure, for patients suffering from dyspnea or acute heart failure at first medical care. Initial heart failure and/or cardiogenic shock are strongly associated with a poorest prognosis.36 Despite this initial difference, the morphine group did not experience significantly more adverse events than the no‐morphine group. The study was not initially designed to evaluate the effect of morphine in patients with anterior STEMI, so the investigation was limited by the number of subjects. It is difficult to evaluate precisely the statistical power of our work. For example, if we previously calculated the number needed to treat with the hypothesis of the 4.2% absolute difference between groups observed in our study, with 80% power and 5% alpha risk, triple sample size would have been necessary. So, limited sample sizes may explain the lack of statistical significance of the observed difference in our work. The use of cyclosporine, even though its known interactions with morphine intestinal absorption and neurological effect, had no significant effect on the results of our study. There was no statistically significant difference between the 4 different subgroups defined by the use of cyclosporine and of morphine, nor significant interaction between morphine and cyclosporine. In the morphine group, we did not record the dose of morphine that was administered as well as corresponding pain scales. Further studies are needed to assess precisely pain management in STEMI patients and its potential impact on clinical outcomes BODY.CONCLUSION: BODY.DISCLOSURES: None. BODY.SUPPORTING INFORMATION: Table S1. Interaction of Morphine With Cyclosporine Effect on MACE Occurrence, Explored by 2‐Way ANOVA With Interaction Term Click here for additional data file.

TITLE: Effectiveness of a Group Support Lifestyle Modification (GSLiM) Programme among Obese Adults in Workplace: A Randomised Controlled TrialGSLiM and Obesity ABSTRACT.BACKGROUND: There was an increasing trend in the prevalence of obesity and its comorbidities over the past decades in Malaysia. Effective intervention for obesity remains limited. This study aimed to compare the effectiveness of a group based lifestyle modification programme amongst obese individuals with an existing dietary counseling programme. ABSTRACT.METHODS: We recruited one hundred and ninety four overweight and obese (BMI>27.5 kg/m2) employees from a local university. They were randomly allocated to either Group Support Lifestyle Modification (GSLiM) (intervention)(n = 97) or dietary counseling (comparison)(n = 97). The GSLIM activities included self monitoring, cognitive-behaviour sessions, exercise as well as dietary change advocacy, which were conducted through seminars and group sessions over 24 weeks. The comparison group was given dietary counselling once in 12 weeks. Both groups were followed up for additional 12 weeks to check for intervention effect sustenance. Anthropometric and biochemical parameters were measured at baseline, 12, 24 and 36 weeks; while dietary intake, physical activities, psychological measures and quality of life measured at baseline, 24 and 36 weeks. Data analysis was conducted using ANOVA repeated measures with intention to treat principle. ABSTRACT.RESULTS: The participants were predominantly women with mean (standard deviation) age of 40.5 (9.3) years. A total of 19.6% of the participants in GSLiM achieved 6% weight loss compared to 4.1% in the comparison group (Risk Ratio 4.75; 95% CI: 1.68, 13.45). At 24 weeks, the retention rate was 83.5% for GSLiM and 82.5% for comparison group. GSLiM participants also achieved significant improvement in total weight self-efficacy score, negative emotions and physical discomfort subscales, MDPSS friend subscale and all domains in quality of life. Participants in the comparison group experienced reduction in negative self-thoughts. ABSTRACT.CONCLUSION: The GSLiM programme proved to be more effective in achieving targeted weight loss, improving weight self-efficacy, friend social support, and quality of life compared to dietary counseling. ABSTRACT.TRIAL REGISTRATION: Iranian Registry of Clinical Trials IRCT201104056127N1 BODY.INTRODUCTION: Malaysia has the highest combined overweight and obese prevalence among the Southeast Asian countries in 2013 [1]. From 1996 to 2009, the prevalence of obesity in Malaysia increased by 23.7% [2], concurrent with the obesity global pandemic. High risks groups for obesity were women, married adults, those with secondary education and unemployed or homemakers [3]. Arguably, working adults who engage in sedentary work as well as those experience stress at the workplace [4,5] are also at risks of obesity. Obesity is associated with increase mortality [6] and reduction in overall quality of life [7]. Malaysians observed an increase of mortality due to heart disease and cancers from 67% in 2008 to 73% in 2012 [8,9]. Comprehensive lifestyle modification on diet, physical activity and psychology remains as the primary intervention for obesity [10,11]. Multicomponent lifestyle modification proved successful in reducing diabetes risk [12] as well as improved cardiovascular risk factors [13]. The Diabetes Prevention Program (DPP) was a high intensity lifestyle modification programme which managed to reduce participant’s diabetic risks by 58% [12]. Yet, in real life, high intensity programme may not be applicable to all settings as it is resource intensive. This results in its translation into the Group Lifestyle Balance (GLB-DPP) implemented in clinical settings, primary care and workplace [14]. The GLB-DPP retained the core principles of the DPP such as 7% weight loss, 150 minutes of physical activity and self-monitoring using group approach with reduced sessions. According to the Social Cognitive Theory; personal behavior, thoughts and environment reciprocate to produce action [15,16]. Factors influencing the cognitive process favoring weight loss includes high self-efficacy [17,18] and social support [19]. Meanwhile, obese individuals may have higher inclination for negative thoughts related to dysfunctional eating [20] hampering lifestyle modification progress. However, these psychological factors were seldom assessed and reported together with clinical measures as part of the lifestyle modification programme outcomes [21,22]. Workplace has been shown to be a feasible and effective setting for lifestyle modification programme for obesity prevention [23–27]. Apart from preventing short term deterioration of quality of life, moderate improvement in weight induced employee productivity [28]. In view of urgent need for obesity intervention, we implemented an adaptation of the GLB-DPP programme named Group Support Lifestyle Modification (GSLiM) in the workplace. The objective was to compare the effectiveness of the GSLiM programme with an existing one to one dietary counseling programme in the workplace. The programme was designed to create social support and improve self-efficacy. It was hypothesized that Group Support Lifestyle Modification (GSLiM) would be more effective in producing weight loss, improvement of cardiovascular risk factors, self-efficacy, social support factors as well as their quality of life. BODY.METHODS: This was a randomised controlled trial conducted in a public university in Kuala Lumpur, Malaysia. Ethical clearance obtained from the Medical Ethics Committee, University Malaya Medical Centre on 16 March 2011 (MEC No. 841.2) and was registered with the Iranian Registry of Clinical Trial (http://www.irct.ir/) (IRCTID: IRCT201104056127N1). There was a slight delayed in trial registration as the authors waited for funding approval obtained from the University Malaya post-graduate research fund. BODY.RECRUITMENT AND PARTICIPANTS: Participants were employees from a public university in Kuala Lumpur. Recruitment started soon after trial registration from May 2011 to September 2012. The trial intervention and follow up commenced from September 2011 to July 2013. Respondents gave written informed consent during recruitment and informed of random allocation into either of the treatment arms. Recruitment, data collection and intervention programme were conducted in the Sport Centre of the Faculty of Sports Science within the university. Eligible criteria included employees of the university, Bahasa Malaysia (national language of the country) literate, with BMI of 27.5kg/m2 or more and able to walk briskly for at least 10 minutes without assistance. Cut off point of 27.5kg/m2 was used as Asians experience higher risk for type 2 diabetes, metabolic syndrome as well as cardiovascular diseases at lower BMI [29–31]. Exclusion criteria included individuals with unstable angina, congestive cardiac failure, cancer, severe pulmonary disease, psychiatry disorders e.g. substance abuse, depression, weight loss of more than 5kg in the last 6 months, pregnant or breastfeeding. Self-administered questionnaire used to assess medical history and family history for disease risks. Further assessment to rule out undiagnosed medical conditions included full blood count (FBC), renal profile (RP), fasting blood sugar (FBS) and lipid profile at recruitment phase. Of 510 employees’ responded to invitations via emails and flyers, 275 consented for participation and assessed for eligibility. Nineteen participants withdrew after consented, eleven did not finish screening, fifty-one were excluded and finally, 194 participants were randomised (Fig 1). 10.1371/journal.pone.0160343.g001Fig 1BODY.CONSORT PARTICIPANTS FLOWCHART OF INTERVENTION AND CONTROL ARM.: GSLiM: Group Support Lifestyle Modification; Comparison group: Dietary counselling. BODY.RANDOMISATION: A third person (TC), who was not involved with the study conducted random allocation of participants into either Group Support Lifestyle Modification (GSLiM) or dietary counseling (comparison). Allocation of participants to GSLiM or comparison arms were assigned using opaque envelopes. This was an open label trial as both treatment provider and participants knew which arm they belonged to since the programme differed between the two groups. BODY.INTERVENTIONS: BODY.GLB-DPP: The Group Lifestyle Balance (GLB-DPP) developed based on the Diabetes Prevention Program (DPP) [32] that retained the core principles of DPP. These included self-monitoring for weight, dietary and physical activity, problem solving, staying motivated, and target 7% weight loss from baseline weight. However, the programme delivery of GLB-DPP used group-approach given over twenty-two hourly sessions in three phases (core, fade frequency and support). BODY.GSLIM: Permission to adopt and adapt the GLB-DPP was obtained from the author/s under the Creative Common Licence Share Alike. The GSLiM programme retained the core characteristics of GLB-DPP except a 6% target weight loss from baseline weight used instead of 7% and absence of support sessions after programme completion at 6th month. The 6% target was based on an achievement of a minimum of 1% weight loss per month in view of evidence that even 5% weight loss may produce clinical improvement among obese individuals [10]. The frequency of sessions conducted in GSLiM was less compared to the GLB-DPP with ten sessions in GSLiM intervention compared to the twenty-two of GLB-DPP. However, the original twenty-two topics of the GLB-DPP were retained in GSLiM with the topics delivered through three seminars and five 90-minute sessions in two phases. The core phase (Phase 1) was run once in two weeks for three months, beginning with a two-day seminar. A fade frequency (Phase 2) started with a one-day seminar, followed by two 90-minute sessions and concluded with a half-day seminar at the end of the active intervention. Comparison of sessions between GLB-DPP and Group Support Lifestyle Modification (GSLiM) is shown in Tables 1 and 2. 10.1371/journal.pone.0160343.t001Table 1BODY.COMPARISON OF GROUP LIFESTYLE BALANCE (GLB) WITH GROUP SUPPORT LIFESTYLE MODIFICATION (GSLIM) PROGRAMME.: Same aspects of GLB and GSLiM Safe and appropriate intervention that incorporates nutrition, physical activity, and behaviour change. Gradual increase of 150 mins per week of physical activity and minus 500 kcal per day of dietary intake. Strong focus on use of self-monitoring tools with feedback. Initial emphasis on fat intake and calories. Primary focus on healthy food choices. Use of inexpensive food samples and incentives. Use of problem-solving techniques to address barriers to healthy eating and physical activity. Group approach. Specific Adaptation to GSLiM GLB Modified GLB (GSLiM) Goal: 7% weight loss and increase physical activity to 150 minutes/week. Goal: 6% weight loss of baseline body weight and physical activity 150 per week. 12 weekly 1-hour sessions delivered over 12–15 weeks 0, 3 and 6 months by seminar sessions. 5 once in 2 weeks session over 12 weeks 2 monthly session over subsequent 12 weeks Pedometer introduced during core sessions Logging for physical activity in log book Trainers: Dietician and exercise specialist Trainers: Dietician and exercise specialist and psychologist Total sessions 22 given in spread of 48 weeks Compressed session to 10 given in 24 weeks 10.1371/journal.pone.0160343.t002Table 2BODY.TOPIC COMPARISON BETWEEN GSLIM AND GLB-DPP.: GLB-DPP topic arrangementGSLiM topic arrangementsCore sessions: Weekly for 12 weeks Core sessions: Bi- weekly (2 per month) for 12 weeks 1. Welcome to GLB. 1. Welcome to GSLiM—Seminar overview of topics. (GLB-DPP 1)Physical activity component (overview of topics and specifics on) Stretching: The truth about flexibility (GLB-DPP session 21) Heart health (GLB-DPP session 20) Diet component: overview of topics, Tip the calorie balance (GLB-DPP 5) Psychological—overview 2. Be a fat and Calorie Detective 2. GSLiM Session 22.1 Fat, calorie and you (GLB-DPP session 2) 2.2 Healthy eating (GLB-DPP session 3) 3. Healthy eating 3. GSLiM Session 33.1 Move those muscles (GLB-DPP session 4) 3.2 Monitoring your activities (GLB-DPP 10) 4. Move those muscles 4. GSliM Session 44.1Negative thoughts and Weight (GLB-DPP 9 and 14) 4.2 Balance your thoughts (GLB-DPP 15) 5. Tip the calorie balance 5. GSLiM Session 5 5.1 Behavior, Environment and Lifestyle Change (GLB-DPP 6 and 17) 5.2 Make Social Cues Work for You (GLB-DPP 11) 6. Take charge of whats around you 6. GSLiM Session 6• 6.0 Strengthen Your Exercise Programme (GLB-DPP 16) 7. Problem solving 8. Four keys to healthy Eating out 9. Slippery slope of Lifestyle change 10. Jump start Your Activity Plan 11. Make Social Cues Work for You 12. Ways to Stay Motivated Transition session (fade frequency) Transition session (fade frequency) 13. Prepare for Long Term Self Management 7. GSLiM Session 7- One day seminar. a. Stress management (GLB-DPP 18). b. Problem solving (GLB-DPP 7). c. Healthy eating out (GLB-DPP 8). d. More volume fewer calories (GLB-DPP 14). e. Group physical activity sessions. 14. More Volume; fewer calories 8. GSLiM Session 8.• 8.0 Standing Up for Your Health (GLB-DPP 19) 15. Balance your thoughts 9. GSLiM Session 9.• 9.0 Stay motivated (GLB-DPP 12) 16. Strengthen Your Exercise Programme 10. GSLiM session 10 (final).Looking back and looking forward (GLB-DPP 22)—Sharing achievements. Prepare long term self-management (GLB-DPP 13) Support sessions (variable sequence) Follow up at 36 weeks 17. Mindful eating 18. Stress and Time Management 19. Standing Up for Your Health 20. Heart Health 21. Stretching: The truth about flexibility 22. Looking back and looking forwards Table 3. 10.1371/journal.pone.0160343.t003Table 3BODY.SUMMARY OF PROGRAMME STRUCTURE, SESSIONS AND CONTENTS.: SessionApproachTitle and Contents1 Seminar Introduction to the programme Foreword on the Group Support Lifestyle Modification (GSLiM) programme configuration and sessions. Highlight energy balance and the concept of self-monitoring. Develop allegiance to the programme. Set up goals for: 6% weight loss, and physical activity of 150 minutes per week. Daily calorie requirement (DCR) minus 500 for dietary intake to be worked out through the 2 step Harris Benedict equation. Foreword on components: psychological, diet and nutrition and physical activity. Description of main food components, sources of fat, food pyramid and food plate theory. General outlook on physical activity and exercise. Introduction to cognitive and behaviour aspects related to lifestyle alteration. Getting-to-know- you session lasting half a day to help participants get acquainted with each other. 2 Group session Calorie measurement and healthy food options. Education and hands on session for self-monitoring through the measurement of food and calories by participants utilising scales and measurement tools. Reading food labels and using the food plate theory for practice. Sessions with active facilitator participation. 3 Group session Moving Your Muscles. Information on effective exercise methods (brisk walking). Raise awareness on the safety aspects of physical activity and exercise. Highlight the enhancement of general health through physical activity and exercise. Assess individual's present level of physical activity and exercise. Rise in activity levels to be spread out over a period of time. Set weekly goals for physical activity. Commence self-monitoring for physical activity and exercise. Group activity for aerobic session. 4 Group session Negative thoughts. Uncover negative thoughts. Increase knowledge on variety of negative thoughts, types of thought disorder and its relationship to behaviour. Suggest ways to offset negative thoughts. Highlight the significance of appreciating oneself for personal accomplishments. Review negative thoughts and set up action plan to counter them. 5 Group session Relationship between behaviour, environment and healthy lifestyle change. Define social and physical environment and relate its influence on physical activity and eating behaviour. Promote positive response to environmental and social drawbacks. Highlight the need for a healthy diet and physical activity /exercise. Emphasize on acting as role models for group members in order to promote physical activity and positive eating. Appraisal of present behaviour and setting up of action plan to achieve positive behaviour change 6 Group session Consolidating Exercise Programme Initiate aerobic fitness sessions and define frequency, intensity, time and type of activity (F.I.T.T). Educate participants on the engagement of self-monitoring to set up an effective exercise schedule through the calculation of heart rate and rate of perceived exertion (RPE). Discuss schedule and include a variety of activities and exercises to develop flexibility, strength, cardiovascular health and endurance. 7 Seminar Consolidate dietary, fitness, thought and behaviour alteration. Report on the progress of participants. Reassess weight loss goals, diet and physical activity based on the most recent measurements. Behaviour: Initiate stress management procedure (abdominal breathing) and problem solving techniques (I.D.E.A.L approach) to deal with issues that crop up due to alterations in diet and daily activities. Dialogue on problems encountered by participants. Encourage healthy eating through the promotion of high volume and low calorie foods. Dietary: Educate on the choice of healthy food when eating out. Explore by group members of ways to be more active on busy work days. Fitness: Suggest activities for work related sitting time. Stress on the need for intervals between extended periods of sitting especially at work. Discuss options on exercise location during unfavourable weather conditions. Teach ways to improve cardiovascular health through resistance and strength exercises. Set up a physical activity schedule for the following month. 8 Group session Challenges in Lifestyle change. Discourse among group members on problems encountered during attempts to change their lifestyles focussing on eating habits and physical activities. Exchange of information and problem-solving suggestions among participants. Re-assess obstacles faced and define a strategy to overcome these barriers to change. 9 Group session Maintaining motivation. Concentrate on means to be constantly motivated for lifestyle change. Stress on awareness of internal and external sources of motivation. Emphasise on appreciating minor accomplishments related to lifestyle change. Initiate discussion among group members on strategy to maintain motivation. 10 Seminar Experience sharing. Disclosure of participants’ accomplishments during the course of the programme (6 months). Initiate panel discussion involving team and group members on the achievements of participants during the programme. Encourage participants to share their six-month experience on the road to better health through weight reduction. Participants were also encourage to continue healthy lifestyle. A starter kit containing handouts, examples of food energy intake and activity-based energy expenditure in the form of wheel chart was provided to participants during the first session. Handouts were also given to participants after subsequent sessions. Self-monitoring activities included weighing-in, submission of diet records and physical activity log was also conducted for each session. Feedback was provided to motivate the participants on each session. BODY.COMPARISON: Participants in the comparison group underwent individual counseling with a dietician once every 12 weeks. Each dietary counseling session lasted for an hour. Recommendations on reduced calorie intake for weight loss, education on food pyramid, self-monitoring of calorie intake, lowering high-fat food and increase of fruits and vegetable intake were given. Participants were advised to increase their physical activity levels and exercise at least three times a week, but no practical session provided. BODY.MEASUREMENTS: BODY.SOCIODEMOGRAPHIC AND LIFESTYLE HISTORY: Sociodemographic characteristics included age, gender, ethnicity, education, household income and marital status; family history of diseases such as hypertension, diabetes and hyperlipidemia; and socially related lifestyle behavior such as smoking and alcohol consumption assessed using self-administered questionnaire. Participants were required to fill a three-day dietary record using a log book. They were taught to estimate food intake with standard day-to-day food utensils. Food items according to meals were entered. A food list with energy (kcal) values was given to assist participants’ in self-monitoring for healthy food choices. Physical activity was measured using the International Physical Activity Questionnaire short form (IPAQ-SF). The IPAQ-SF comprised of seven items reported in terms of METS-min per week which can be summarised into three main categories (low, moderate and high). It is a valid measurement of physical activity and has moderate to high reliability [33]. BODY.ANTHROPOMETRY, BIOCHEMICAL AND CLINICAL MEASURE: Weight was measured using SECA digital scale (Model HD 309) and height was measured using SECA body meter (Model 208) to the precision of 0.1 kg and 0.1 cm, respectively. Participants wore light clothing, and stood on scale without shoes with leg apart within the scale footprint. Waist and hip circumference were measured using non elastic SECA measuring tape to the nearest 0.1 cm following the WHO guideline [34]. Digital OMRON Model HEM 907 was used to measure blood pressure based on the Malaysian Clinical Practice Guidelines for management of Hypertension [35]. Fasting blood samples (after 8 hours) collected by trained staff and sent within four hours of collection in an icebox to the laboratory. Measurements were conducted by trained research assistants at baseline, 12, 24 and 36 weeks. Height measured only once at baseline. BMI was calculated as weight in kilograms divided by height in meters squared. BODY.PSYCHOLOGICAL MEASURES AND QUALITY OF LIFE: Psychological measures and quality of life were measured using self-administered questionnaires at baseline, 24 and 36 weeks. Weight efficacy lifestyle (WEL) questionnaire was used to measure eating self-efficacy in weight management with 20 items in five subscales. The subscales include food control in negative emotions, availability of food, social pressure, physical discomfort and physical activity with internal consistency ranged from 0.7 to 0.9 [36]. The Malay version of WEL questionnaire had internal consistency (Cronbach’s alpha) of 0.47 to 0.86 [37]. Social support was measured using the Multidimensional Perceived Social Support (MDPSS) which comprised of 12 items and three subscales. Internal reliability achieved was 0.82 for perceived social support from family, 0.88 for friends and 0.94 for significant others [38]. The Malay version of MDPSS has internal consistency of 0.89 with reliability coefficients for family, friends and significant others subscales ranging from 0.67 to 0.83 [39]. Automatic Thought Questionnaire (ATQ) used to assess negative thoughts [40]. Due to poor construct, the Malay version was reduced to 17 items and two subscales from 30 items [41]. Each subscale i.e. negative self concept (NSE) and personal maladjustment (PMA) had internal consistency (Cronbach alpha) of 0.91 and 0.83 respectively. Method of scoring of the Malay ATQ remained the same as the original 30 item with a cut-off score of 30 for depression. Quality of life was measured using the WHO Quality of Life short form questionnaire (WHOQoL-BREF), a shorter version of WHO Quality of Life (WHOQoL) questionnaire with the objective to measure a global perspective of quality of life across cultures [42]. The WHOQoL-BREF comprises of 26 questions in four domains i.e. physical activity, psychological, social support and environment. Validation of the translated Malay version revealed high reliability with internal consistency of 0.64–0.80, and test—retest-reliability of 0.49–0.88 [43,44]. BODY.SAMPLE SIZE: Sample size was calculated using OpenEpi software. Based on Renjilian et al.’s findings (17), 49% of group therapy participants achieved ten percent weight loss compared to 29% in individual counseling. A total of 286 participants was required after setting the study power at 80%, level of significance at p<0.05 and attrition rate of 25%. Due to low response rate, high number of excluded respondents and time constraint for further recruitment, the sample size was not achieved. Power calculation revealed the sample size of 194 achieved 88.8% power for targeted weight loss. BODY.STATISTICAL ANALYSIS: IBM SPSS Statistics, Version 20 for Windows (SPSS Inc, Chicago IL) used for data analysis. The dietary data was cross-checked and entered by a trained dietician into Nutritionist Pro where the Malaysian nutrient database was used. Between group mean differences at baseline for continuous variables were analyzed using t-tests and χ2 tests for categorical data. Mean differences between groups over time used analysis of variance for repeated measures. All analysis was carried out using the intention to treat principles. Missing data was imputed using the last observation carried forward. Continuous data at baseline was described as means± standard deviation. Repeated measure data for within group measures was described as mean ±standard error. Confidence interval (CI) was reported where appropriate. Statistical significance was preset at 0.05. Relative risk reported for proportion achieved for weight loss, while Cohen’s d used for the within and between group repeated measure. Adjustment conducted for measures where the baseline values showed significant between-group differences to control for bias i.e. carbohydrate percentage and multidimensional perceived of social support. BODY.RESULTS: BODY.BASELINE CHARACTERISTICS: A total of 194 participants recruited and randomised. The participants’ mean age (standard deviation) was 40.5 (9.3) years. Majority of participants were women (72.7%) and non-academician (92.3%). More than half (64%) had at least secondary education. A total of 81 (83.5%) GSLiM participants and 80 (82.5%) of dietary counseling participants completed the study at 24 weeks (Fig 1). Participants from the GSLiM group had higher total MDPSS score, family support and friend subscales score as well as carbohydrate percentage intake. No other significant difference between groups was observed at baseline (Tables 4 and 5). 10.1371/journal.pone.0160343.t004Table 4BODY.BASELINE MEASURES.: GSLiM, mean (sd), n = 97Diet counselling, mean (sd), n = 97p valueAge, years 39.7 (9.2) 40.4 (9.5) 0.617 Gender 0.629 Female, n (%) 69 (71.1) 72 (74.2) Male, n (%) 28 (28.9) 25 (25.8) Ethnicity 0.296 Malay, n (%) 87 (89.7) 91 (93.8) Non Malays, n (%) 10 (10.3) 6 (6.2) Education, tertiary, n (%) 39 (40.2) 29 (29.9) 0.132 Household income (RM) 2680.34 (1511) 2504.19 (1641.20) 0.438 Married, n. (%) 76 (78.4) 74 (76.3) 0.732 Occupation, academician, n (%) 9 (9.3) 6 (6.2) 0.420 Family history of: Hypertensive, n (%) 11 (11.3) 13 (13.4) 0.663 Diabetes, n (%) 3 (3.1) 2 (2.1) 0.683 Hyperlipidaemia, n (%) 8 (8.2) 13 (13.4) 0.248 Behaviour: Non-alcohol intake, 96 (99) 97 (100) 0.316 Smoker, n (%) 9 (9.3) 5 (5.2) 0.267 Height, m 1.59 (0.08) 1.58 (0.09) 0.863 Weight, kg 82.3 (16.3) 82.0 (13.5) 0.911 BMI, kg/m2 32.4 (4.8) 32.4 (3.8) 0.980 Waist circumference, cm 95.1 (11.2) 94.9 (9.5) 0.872 Hip circumference, cm 112.2 (10.4) 111.1 (8.2) 0.429 Systolic blood pressure, (mmHg) 128.6 (16.1) 129.1 (14.4) 0.712 Diastolic blood pressure, (mmHg) 83.0 (12.2) 83.4 (10.7) 0.774 Triglyceride, (mmol/l) 1.41 (0.62) 1.56 (0.82) 0.182 Total Cholesterol, (mmol/l) 5.2 (0.9) 5.3 (1.1) 0.913 HDL Cholesterol, (mmol/l) 1.23 (0.29) 1.25 (0.31) 0.592 LDL Cholesterol, (mmol/l) 3.45 (0.82) 3.35 (0.85) 0.426 IPAQ SF total METmin-1 per week, 1071 (3.24) 1318.3 (3.06) 0.238 Weight efficacy lifestyle 123.0 (25.6) 121.6 (23.4) 0.697 Negative emotions 25.6 (6.5) 25.1 (6.3) 0.560 Availability 21.6 (6.6) 21.4 (6.1) 0.901 Social Pressure 22.3 (6.1) 22.1 (5.7) 0.771 Physical Discomfort 27.7 (5.3) 26.9 (5.6) 0.315 Positive activities 25.8 (5.7) 26.1 (5.2) 0.693 Multidimensional social support a 66.3 (9.9) 62.8(8.7) 0.010b Family Support 22.8 (3.7) 21.7 (3.3) 0.029c Friend Support 20.5 (3.9) 19.3 (3.4) 0.021c Significant Others 22.9 (4.1) 21.8 (4.5) 0.061 Total automatic thoughts questionnaire score 26.5 (7.4) 26.7 (8.0) 0.867 Negative self-concept 16.1 (4.6) 16.2 (4.6) 0.853 Personal mal-adjustment 9.1 (2.8) 9.2 (3.5) 0.822 WHOQoL BREF Physical Health 12.9 (1.6) 13.1(1.4) 0.373 Psychological 13.1 (1.7) 13.1 (1.7) 0.942 Social Relationship 14.8 (2.7) 14.6 (2.2) 0.681 Environment 14.1 (1.8) 14.0 (1.9) 0.722 a Significant p<0.001. b Significant p<0.01. c Significant p<0.05. 10.1371/journal.pone.0160343.t005Table 5BODY.BASELINE DIETARY INTAKE.: GSLiM, mean (sd) N = 65Diet counselling, mean (sd), n = 56p valueDietary energy intake, mean kcal/day 1421.2 (332.3) 1351.3 (369.8) 0.276 Protein (%) 15.8 (2.3) 15.0 (2.8) 0.109 Carbohydrate (%) 53.3 (6.5) 55.8 (6.1) 0.034a Fat (%) 30.8 (5.4) 29.0 (5.1) 0.064 Cholesterol (mg) 168.0 (88.6) 162.3 (107.2) 0.748 Saturated Fat (mg) 8.1 (3.9) 9.0 (4.5) 0.201 Mono saturated fat (mg) 7.3 (3.6) 7.2 (3.0) 0.772 Meals frequency 3.4 (0.6) 3.6 (0.7) 0.068 a Significant p<0.05. BODY.BETWEEN GROUP DIFFERENCES: At week 24, nineteen participants (19.6%) achieved 6% targeted weight loss in the intervention compared to 4 (4.1%) in the comparison group, Risk Ratio (RR) of 4.75 (95% CI: 1.68, 13.45). Out of 23 participants achieved weight loss, 10 (57.5%) intervention participants and 3 (75%) from the comparison group retained the targeted weight loss (6%) at 36th week. However, when measured in terms of weight in kg, no significant difference observed for the mean difference in weight loss between groups (-0.82 ± 2.12 kg), Cohen’s d 0.001. Table 6 summarises obesity and cardiovascular measures from baseline to 12th week, 13th to 24th week, baseline to 24th week and from 25th week to 36th week. No other significant mean difference between groups observed at any of the time intervals. Physical activity, psychological and quality of life scores measure from baseline to 24th week and from 25th week to 36th week are shown in Table 7. 10.1371/journal.pone.0160343.t006Table 6BODY.OBESITY AND CARDIOVASCULAR OUTCOMES BETWEEN AND WITHIN GROUPS OVER TIME.: Within GroupBetween groupsGSLiM mean change (s.e.) N = 97Comparison mean change (s.e.) N = 97Mean difference (95% CI)Cohen’s dp value0 to 12 weeks (intensive phase) Weight (kg) -1.52 (0.36)a -0.09(0.19) - 0.47 (4.69, 3.74) 0.000 0.825 BMI (kg/m2) - 0.60 (0.14)a -0.31(0.08) - 0.30 (-1.52,0.92) 0.001 0.627 Waist circumference (cm) - 1.67 (0.48)a 0.54 (0.39) - 0.87 (-3.79, 2.06) 0.002 0.561 Hip circumference (cm) -0.74(0.39) -0.16 (0.27) 0.77 (-1.81, 3.35) 0.002 0.557 SBP (mmHg) 0.24 (1.19) -2.26 (1.01) 0.43 (-3.59, 4.46) 0.000 0.832 DBP (mmHg) -0.25(0.96) -1.38 (0.72) 0.09 (-2.91, 3.09) 0.000 0.951 Fasting serum triglyceride (mmHg) 0.02 (0.05) 0.009 (0.047) -0.14 (-0.33, 0.07) 0.009 0.184 Fasting serum cholesterol (mmHg) -0.04 (0.06) -0.04 (0.05) -0.01 (-0.26, 0.23) 0.000 0.913 HDL (mmHg) -0.031 (0.017) 0.018 (0.016) -0.05 (-0.13, 0.04) 0.007 0.256 LDL (mmHg) -0.058 (0.055) -0.049 (0.057) 0.09 (-0.12, 0.31) 0.004 0.401 Fasting blood glucose (mmHg) -0.003 (0.086) 0.121 (0.087) -0.11 (-0.39, 0.17) 0.003 0.442 13 to 24 weeks (fade frequency) Weight (kg) -0.92 (0.26)b -0.61(0.22)b -1.35 (-5.54, 2.85) 0.002 0.527 BMI (kg/m2) -0.36 (0.10)b -0.24 (0.08) -0.64 (-1.87, 0.58) 0.006 0.300 Waist circumference (cm) 0.16 (0.44) 0.18 (0.37) -1.79 (-4.73, 1.14) 0.008 0.229 Hip circumference (cm) -0.44 (0.29) -0.27 (0.23) 0.39 (-2.21, 3.00) 0.000 0.763 SBP (mmHg) 0.17 (1.08) 0.27 (0.90) 1.63 (-2.37, 5.65) 0.003 0.424 DBP (mmHg) -1.07 (0.82) 1.27 (0.85) -0.51 (-3.51, 2.49) 0.001 0.737 Fasting serum triglyceride (mmHg) -0.05 (0.05) -0.047 (0.042) 0.01 (-0.24, 0.37) 0.007 0.911 Fasting serum cholesterol (mmHg) 0.08 (0.05) -0.02 (0.04) -0.13 (-0.35, 0.09) 0.000 0.239 HDL (mmHg) 0.038 (0.018) 0(0.013) -0.05 (-0.14, 0.03) 0.008 0.215 LDL (mmHg) 0.033(0.049) 0.039(0.041) 0.08 (-0.4, 0.302) 0.003 0.453 Fasting blood glucose (mmHg) -0.088(0.046) -0.070(0.117) -0.21 (-0.54, 0.12) 0.008 0.207 0 to 24 weeks Weight (kg) -2.42 (0.49)a -0.69 (0.27)c -0.82 (-5.00, 3.37) 0.001 0.701 BMI (kg/m2) -0.96 (0.19)a -0.27 (0.11)b -0.44 (-1.65, 0.78) 0.003 0.480 Waist circumference (cm) -1.51(0.52)a -0.31 (0.12) -1.12 (-4.00, 1.77) 0.003 0.445 Hip circumference (cm) -1.18 (0.45)c -0.43 (0.34) 0.62 (-1.96, 3.19) 0.000 0.850 SBP (mmHg) 0.41 (1.23) -1.99 (1.14) 0.82 (-3.09, 4.72) 0.000 0.680 DBP (mmHg) -1.32 (0.91) -0.11 (0.99) -0.49 (-3.4.1, 2.41) 0.003 0.736 Fasting serum triglyceride (mmHg) -0.03 (0.06) -0.04 (0.05) -0.13 (-0.34, 0.07) 0.009 0.199 Fasting serum cholesterol (mmHg) 0.04 (0.07) -0.02 (0.06) 0.005(-0.24, 0.25) 0.000 0.970 HDL (mmHg) 0.01 (0.02) 0.02 (0.02) -0.04 (-0.13, 0.04) 0.002 0.302 LDL (mmHg) -0.03 (0.07) -0.01 (0.06) 0.09 (-0.13, 0.30) 0.003 0.418 Fasting blood glucose (mmHg) -0.09 (0.08) 0.11 (0.09) -0.16 (-0.45, 0.13) 0.005 0.299 25–36 weeks (follow-up) Weight (kg) 0.19 (0.35) -0.11 (0.16) -1.42 (-5.65, 2.82) 0.002 0.510 BMI (kg/m2) 0.02(0.07) -0.04 (0.64) -0.67 (-1.92, 0.57) 0.006 0.286 Waist circumference (cm) 0.19 (0.35) -0.19 (0.37) -1.44 (-4.39, 1.52) 0.005 0.338 Hip circumference (cm) -0.45 (0.31) -0.33 (0.21) 0.26 (-2.39, 2.91) 0.001 0.636 SBP (mmHg) -1.70 (1.27) 0.46 (0.90) 0.51 (-3.57, 4.48) 0.000 0.802 DBP (mmHg) -0.72(0.97) -1.38(0.78) -1.03 (-4.08, 2.03) 0.002 0.509 Fasting serum triglyceride (mmHg) 0.008(0.04) -0.01(0.03) 0.008 (-0.25, 0.26) 0.007 0.264 Fasting serum cholesterol (mmHg) 0.01(0.06) 0.07(0.04) -0.13 (-0.36, 0.09) 0.000 0.953 HDL (mmHg) 0.012 (0.015) 0.003 (0.018) -0.02 (-0.07, 0.11) 0.003 0.489 LDL (mmHg) -0.033 (0.063) 0.067 (0.044) 0.03 (-0.19, 0.26) 0.001 0.715 Fasting blood glucose (mmHg) -0.008 (0.048) -0.065 (0.039) -0.22 (-0.58, 0.14) 0.006 0.267 CI, confidence interval; GSLiM, Group Support Lifestyle Modification. a Significant p<0.001. b Significant p<0.01. c Significant p<0.05. All participants with initial weight measurement were included in this intention to treat analysis. From analysis of repeated measures treatment against time with time as the dependent variable. Effect size stated using Cohen’s for between group mean differences. 10.1371/journal.pone.0160343.t007Table 7BODY.PHYSICAL ACTIVITY, PSYCHOLOGICAL AND QUALITY OF LIFE SCORE BETWEEN AND WITHIN GROUPS OVER TIME.: Within groupBetween groupsIntervention group mean change (s.e.)(n = 97)Control group mean change (s.e.)(n = 97)Mean Difference at intervals (95% CI)Cohen dP0 to 24 weeks Total IPAQ SF Score, mets min-1 (sd)* -18.9 (236.5) 90.3(98.2) -244.3(-790.1, 301.5) 0.004 0.378 Vigorous, mets min-1 -75.5 (149.7) 58.6 (63.9) -10.5(-296.4, 275.5) 0.000 0.943 Moderate, mets min-1 -6.8 (95.9) 50.9 (59.5) -238.6(-509.3, 32.2) 0.015 0.084 Walking, mets min-1 63.3 (79.6) -19.3(43.8) 4.7(-212.0,221.5) 0.000 0.966 Total WEL score 10.3 (2.46)a 1.78(1.88) 5.62 (-0.10, 11.35) 0.019 0.054 Negative emotions, mean (sd) 2.02 (0.57)b 0.07 (0.47) 1.51 (0.004, 3.02)c 0.020 0.049 Availability, mean (sd) 2.63 (0.62)a 0.93 (0.49) 0.96 (-0.58, 2.50) 0.008 0.218 Social Pressure, mean (sd) 1.71 (0.60)c 0.52 (0.43) 0.85 (-0.61, 2.29) 0.007 0.253 Physical Discomfort, mean (sd) 1.41 (0.48)c 0.36 (0.44) 1.31 (0.05, 2.56)c 0.022 0.041 Positive activities, mean (sd) 2.52(0.54)a -0.09 (0.40) 0.99 (-0.27, 2.26) 0.012 0.121 # Total MDPSS mean score 1.46 (0.65) 1.09(0.63) 0.012 0.127 # Friend Support, mean (sd) 0.86(0.25)b 0.44 (0.22) 0.41(0.08, 0.75)a 0.030 0.015 #Family Support, mean (sd) 0.10 (0.27) 0.19 (0.25) 0.09 (- 0.25, 0.44) 0.002 0.578 #Significant Others, Mean (sd) 0.50 (0.30) 0.45 (0.32) 0.19(-0.21, 0.59) 0.005 0.347 Total ATQ mean score -0.84 (0.68) -0.40 (0.43) -0.40 (-2.29, 1.49) 0.001 0.675 Negative self-concept mean score -0.39 (0.45) -0.26 (0.25) -0.19 (-1.35, 0.97) 0.001 0.745 Personal maladjustment mean score -0.36 (0.26) -0.19 (0.23) -0.19(-0.94, 0.57) 0.001 0.630 WHOQOL BREF Physical health 2.35(0.19)a 1.82(0.19) a 0.07(-0.35, 0.48) 0.001 0.743 Psychological 1.62(0.15) a 1.52(0.16) a 0.06(-0.41, 0.52) 0.000 0.784 Social relationship 0.63(0.22)c 0.12 (0.17) 0.39(-0.22, 1.01) 0.008 0.210 Environment 0.52(0.14)b 0.25(0.11)b 0.18(-0.31, 0.67) 0.003 0.471 25 to 36 weeks (follow up) Total IPAQ SF Score, mets min-1 (sd)* 1.1(143.6) 303.1(138.4) -449.9 (-1015.1,115.3) 0.013 0.118 Vigorous, mets min-1 -63.1 (73.2) 59.4 (79.6) -138.8 (-437.5, 159.9) 0.004 0.361 Moderate, mets min-1 62.5 (61.5) 102.1 (82.0) -287.2 (-563.2, -11.2)c 0.021 0.041 Walking, mets min-1 1.7 (73.4) 141.6 (63.6) - 23.9 (-240.8, 192.9) 0.000 0.828 Total WEL score -1.77 (1.45) 0.45 (1.19) 8.76 (3.05, 17.47)b 0.045 0.003 Negative emotions, mean (sd) -0.42 (0.39) 0.06 (0.27) 2.24 (0.81, 3.67)b 0.305 0.002 Availability, mean (sd) -0.09 (0.35) 0.19 (0.28) 1.68 (0.10, 3.25)c 0.022 0.037 Social Pressure, mean (sd) -0.34 (0.34) 0.09 (0.26) 1.23 (-0.29, 2.75) 0.013 0.114 Physical Discomfort, mean (sd) -0.40 (0.31) 0.31 (0.25) 1.48 (0.28, 2.68)c 0.030 0.016 Positive activities, mean (sd) -0.52 (0.36) -0.79 (0.33) 2.14 (0.93, 3.35)a 0.060 0.001 #Total MDPSS mean score 0.35(0.62) 0.46(0.41) 1.46 (-0.26, 3.17) 0.015 0.095 # Friend Support, mean (sd) -0.10(0.19) 0.11(0.14) 0.80 (0.15, 1.45) c 0.030 0.017 #Family Support, mean (sd) 0.27(0.22) 0.16(0.18) 0.29 (-0.35, 0.95) 0.004 0.364 #Significant Others, Mean (sd) 0.09(0.31) 0.19(0.18) 0.38 (-0.38, 1.13) 0.005 0.325 Total ATQ mean score -0.19(0.47) -0.73(0.21)b -0.35 (-2.17, 1.48) 0.001 0.709 Negative self-concept mean score -0.10(0.30) -0.47(0.37)b -0.07 (-1.22, 1.07) 0.000 0.901 Personal maladjustment mean score -0.08(0.19) -0.19 (0.09) -0.21 (-0.94, 0.52) 0.002 0.568 WHOQOL BREF Physical health -0.92 (0.18)a -0.53(0.19)c 0.13(-0.37, 0.64) 0.001 0.609 Psychological -0.29 (0.19) -0.12 (0.20) 0.03(-0.49, 0.54) 0.000 0.923 Social relationship -0.03 (0.17) 0.14 (0.11) 0.56(-0.08, 1.19) 0.015 0.084 Environment -0.06 (0.11) 0.06 (0.09) 0.21(-0.28, 0.69) 0.004 0.405 CI, confidence interval; GSLiM, Group Support Lifestyle Modification. a Significant p<0.001. b Significant p<0.01. c Significant p<0.05. All participants with initial weight measurement were included in this intention to treat analysis. From analysis of repeated measures treatment against time with time as the dependent variable. Effect size stated using Cohen’s for between group mean differences. # MDPSS, adjusted for baseline MDPSS. * median values instead of mean for physical activity. The intervention group achieved higher score for negative emotions and physical discomfort of WEL subscales compared to the comparison group from baseline to 24th week. The effects maintained during follow up from 25th to 36th week. Intervention participants achieved higher friend support mean score from baseline to 24th week, even after adjusting for baseline MDPSS score. During the follow up period, except for social pressure, total WEL and other WEL subscales mean score increased significantly compared to the comparison group. However, moderate physical activity in METSmin-1 per week reduced significantly in the intervention group compared to the comparison group during 25th to 36th week. The intervention group experienced lower intake in carbohydrate percentage with higher percentage increase in fat intake over baseline to 12th week and 13th to 24th week. No other dietary change was observed (Table 8). 10.1371/journal.pone.0160343.t008Table 8BODY.DIETARY ENERGY INTAKE BETWEEN AND WITHIN GROUPS OVER TIME.: Within group changeBetween groupsIntervention mean (s.e)Control mean (s.e)Mean difference at intervalsCohen dPN = 65N = 56(95% CI)0–12 weeks Dietary intake, kcal 0.41 (38.2) 45.70 (22.76) 47.25 (-79.8, 174.3) 0.005 0.463 Protein (%) -0.05 (0.29) -0.19 (0.20) 0.88 (-0.01, 1.77) 0.09 0.053 Carbohydrate (%)# -4.12 (0.82)a -1.58 (0.67) -1.78 (-2.86, -0.69)b 0.08 0.002 Fat (%) 3.54 (0.93)b 1.77 (0.66) 2.69 (0.91, 4.47)b 0.07 0.003 13–24 weeks Dietary intake, kcal 5.06 (12.96) 15.78 (35.53) 19.24 (-113.3, 151.8) 0.001 0.744 Protein (%) 0.08 (0.16) 0.22 (0.24) 0.93 (-0.02, 1.87) 0.03 0.055 Carbohydrate (%)# -0.19 (0.40) -1.91 (0.51)b -2.76 (-4.87, -0.64)c 0.05 0.011 Fat (%) 0.11 (0.35) 2.02 (0.53)b 2.62 (0.36, 4.88)c 0.04 0.023 25–36 weeks Dietary intake, kcal -6.52 (9.08) 39.15 (23.7) -8.96 (-139.7, 121.8) 0.00 0.892 Protein (%) 0.12 (0.09) 0.44 (0.19) 0.69 (-0.30, 1.69) 0.02 0.171 Carbohydrate (%)# -1.18 (0.13) -1.48 (0.62) -1.33(-3.61, 0.95) 0.11 0.250 Fat (%) -0.07 (0.11) 1.05 (0.55) 1.17 (-1.16, 3.51) 0.01 0.322 0–24 weeks Dietary intake, kcal 5.46 (38.28) 61.48 (36.17) 36.12 (-86.9, 159.19) 0.003 0.562 Protein (%) 0.13 (0.30) 0.03 (0.20) 0.81 (-0.08, 1.68) 0.001 0.444 Carbohydrate (%)# -4.31(0.88)a -3.49 (0.64)a -1.84(-3.25, -0.43)c 0.053 0.011 Fat (%) 3.65 (0.96)b 3.79 (0.82) 1.74 (0.003, 3.46) 0.000 0.050 Confidence interval; GSLiM, Group Support Lifestyle Modification. a Significant p<0.001. b Significant p<0.01. c Significant p<0.05. All participants with initial weight measurement were included in this intention to treat analysis. From analysis of repeated measures treatment against time with time as the dependent variable. Effect size stated using Cohen’s for between group mean differences. # CHO percentage was adjusted for its baseline value for between group analyses in all interval. BODY.WITHIN INTERVENTION GROUP: Weight and BMI of the intervention group were reduced during the first 12 weeks (core phase), with smaller reduction in the next 12 weeks (fade frequency). During follow up, there was an insignificant increase in weight and BMI. Reduction in waist circumference occurred in the first 12 weeks but not sustained during fade frequency. An overall significant hip circumference reduction was observed within interval 0 to 24th week, although the reduction was not significant in the core phase or fade frequency. WEL and its subscales’ scores, friend support subscale score of MDPSS and WHOQOL-BREF domains score increased from 0 to 24th week. However, the improvement was not sustained during follow up, with significant reduction in WHOQOL-BREF physical health score observed during 25th to 36th week. The intervention group experienced reduction in carbohydrate percentage intake mainly during the core phase, resulting in significant reduction from baseline to 24th week. Concurrent fat percentage intake increased during the same time interval. There was no significant change in cardiovascular risks and physical activity measure throughout the study. BODY.WITHIN COMPARISON GROUP: The comparison group weight decreased only during 13th to 24th week, yet it contributed to the overall significant reduction throughout treatment from 0 to 24th week. During the same 0 to 24th Significant decreased in carbohydrate and increased in fat percentage intake, occurred from 13th to 24th week, in the comparison group. As per intervention group, the WHOQOL-BREF physical health score decreased in the follow up phase. Apart from these changes, significant reduction was also observed in negative thoughts and the negative self-concept subscale of the ATQ score during follow up period (25th to 36th weeks). BODY.ADHERENCE TO INTERVENTION: BODY.ATTENDANCE: Attendance was assessed as part of adherence measure in the intervention group. Of the total six sessions within the first 12 weeks, 33 (17.0%) participants in the intervention group attended more than four sessions, 34 (17.5%) attended three to four sessions while 30 (15.5%) attended two sessions and less. During the fade frequency with four sessions, 20.6 percent participants attended three to four sessions. None of the participants who attended less than three sessions achieved 6% weight loss, while 8.9% of participants attended four to six sessions achieved the targeted weight loss. About half (44.8%) of the participants attended more than seven sessions achieved 6% targeted weight loss (p<0.001). Median attendance was six sessions with inter-quartile range of five sessions. There was positive correlation between attendance and weight loss (r = 0.491, P<0.001). BODY.LOG BOOK SUBMISSION: The overall log book submission was unsatisfactory for both groups. Only 46.4% (n = 45) intervention participants submitted their log books at 12th week and 16.5% (n = 16) at 24th week. Only 23.7% (23) comparison group participants submitted their log books at 12 weeks and 19.6% (19) at 24th week. Those who did not submit their log books were of younger age (p = 0.03). BODY.ADVERSE EFFECT OF THE INTERVENTION: Two male participants with BMI above 40kg/m2 from the intervention group experienced mild soft tissue injury during self-conducted exercise and had undergone treatment. No other serious event reported. BODY.DISCUSSION: The intervention proved to be more effective in achieving the targeted 6% weight loss, improved self-efficacy in dietary control, and achieved better friend support and quality of life than the comparison (dietary counseling) group. The psychological improvement in the intervention group was sustained post intervention. Although the comparison group also experienced reduction in weight, BMI, improvement in quality of life, and ATQ, there was no improvement in self-efficacy and social support. A total of 19% of GSLiM participants achieved 6% weight loss. For comparison purpose, we recalculated and found 17.5% of GSLiM participants achieved 7% weight loss. Other GLB based study reported 23 to 37% achievement in targeted weight loss [14]. A most recent GLB based trial on work site reported 29% of participants achieved targeted 7% weight loss [26]. While another study assessed the effectiveness of the coach led GLB programme in primary care setting had 37% participants achieving 7% weight loss at 15 months [45]. Our study had lower proportion of targeted weight loss due to fewer sessions in GSLiM compared to the original GLB-DPP. Earlier study reported for every other lifestyle session attended, weight loss could increase by 0.26% (16). Our findings concurred with recent review which showed multicomponent behaviour intervention was more effective than single component intervention [46]. The mean weight loss within intervention at 24th week was -2.43 kg (95% CI: -3.75, -1.21, p = 0.001), comparable to another trial which reported mean weight loss of -2.3 kg (95% CI: -2.92 to -1.72, p = 0.001) [47]. Most improvement occured during the first 12 weeks in the intervention group where sessions were more intensive. Earlier findings showed positive association between intensity and magnitude of weight loss [48]. Yet, lower intensity programme with similar effect is still being sought by other researchers [49], as intensive intervention are costly in terms of manpower and materials [50]. Apart from weight loss, significant improvement of the self-efficacy score and its subscales were seen among intervention participants compared to the comparison group, similarly found by other studies [51–53]. The intervention group experienced immediate improvement in social support score at baseline. However, after controlling for the baseline friend support score, significant difference between groups was observed for mean friend support score during the 24 weeks. Similar finding was also reported by a recent cluster randomised trial where healthy eating was found to be supported by friend [54]. Any improved ATQ score would be expected in the intervention group due to the presence of cognitive elements incorporated in the programme, instead the improvement of ATQ score occurred within the comparison group. The use of ATQ to measure negative thought in obesity research remains limited in the presence of other tools used to assess dysfunctional eating behavior associated with obesity [55]. The average cut off value for depressogenic negative thoughts using the 17 item scale of ATQ—Malay in non-clinical population was 30.0 and clinical population was 47.0 [41]. The mean ATQ score for both the intervention and comparison groups was 25.6 and 26.3 respectively, below the cut off value; therefor the significant mean difference found between the intervention and comparison group was most likely due to chance. ATQ was used in our study to measure depressogenic features amongst obese participants and observed further change as treatment progressed. Moderate concurrent validity (r = 0.65) was found between the Malay ATQ and Malay-Beck Depression inventory (BDI), while discriminant analysis achieved 89.3%. Other than improvement in weight, psychological measures and quality of life, there was no improvement in cardiovascular risks among our participants. Other studies found at least 10% weight loss of baseline weight resulted in changes for the cardiovascular risk parameters [56]. Several other reasons may influence the absence of treatment effect on cardiovascular risks in this study. Our participants were within the younger age group and their clinical indicators were of normal range, therefor further improvement would be less likely. The focus of intervention would also influence the result as other studies targeting clinical indicators with health education in dietary approach managed to result in cardiovascular risks improvement [57,58]. Therefore, weight loss may not necessary resulting improvement of cardiovascular risks factors. Participants also experienced small reduction in carbohydrate percentage intake and increase in fat percentage intake, as found by previous study [59]. Low carbohydrate diet has been associated with greater weight loss compared to low fat diet [60]. Nevertheless, there has been call that the effectiveness of lifestyle modification not to be overshadowed by the continuous search for the best dietary approach [61] when moderate balanced nutrients intake should be advocated. Although promising, the results of this study should be interpreted cautiously. Since participants were employees from the university, generalizability of the study remained limited within population of the same setting and may differ with the general population. Self—reported measures in particular self-administered surveys used may result in reporting bias. Negative behavior such as smoking and alcohol consumption may result in underreporting, while physical activity may be over reported. Although the short form IPAQ was advocated to be used in view of time-saving, a recent review [62] found that IPAQ SF tended to overestimate the METs-minutes/ week score. However, the categories of physical activity derived from the long form IPAQ [63] showed similarity with the categories found in our study and another Malaysian based study [3]. Therefore, we foresee the results to be reliable in particular in assessing change over time. Objective measures such as pedometer or accelerometer cited to be more reliable [64] however its utilization is resource consuming and may not be feasible in community setting. Our study only measured dietary self-efficacy, however, recent finding showed that exercise self-efficacy may be a better predictor for weight loss compared to dietary self-efficacy [65]. Considering the continuous development in the cognitive- psychological aspect of obesity intervention, both physical and dietary self-efficacy need to be assessed in future studies. Self-monitoring is another critical component for weight reduction (39), however the log book submission by both groups was poor although attempts had been made to improve submission. This may further explain the small effect size observed and no improvement in cardiovascular risks although weight loss was achieved within each group. Poor log book submission for physical activity and dietary intake has also been reported in other studies (17). The attendance for group sessions was low compared to other studies [25,45,66]. Yet similar findings to ours was also found in another Malaysian based lifestyle intervention trial [67]. We also found processes such as attendance and log book submission to be correlated with weight loss. Competing time work demand has also been cited as factors affecting attendance for work site intervention [25]. Local culture within the society may influence the attendance towards programme which needs to be explored. Finally, we did not evaluate environmental support such as availability of healthy food choices and physical activity facilities which is known to influence weight loss. Nevertheless, this study is the first group based programme derived from the GLB-DPP conducted in Malaysia. It addressed the dynamics of psychological aspect within the lifestyle modification programme based on a theoretical construct which was rarely implemented and monitored in other translational weight loss programme. The randomised controlled design as opposed to single arm trial was used to evaluate its effectiveness compared to an existing dietary programme. BODY.CONCLUSION AND RECOMMENDATIONS: Our findings showed the group based workplace intervention (GSLiM) programme managed to achieve targeted weight loss, improved self-efficacy and created positive support with lower intensity. The incorporation of group based approach and psychological sessions managed to achieve psychological change needed to achieve weight loss. The GSLiM programme is ready to be used and can be replicated in similar setting with possible enhancement for exercise self-efficacy, attendance, and adherence to self-monitoring. The programme should be extended for longer duration. Further research should explore the predictive value of the psychological factors in assisting participants to achieve their targeted weight loss. BODY.SUPPORTING INFORMATION: S1 FileBODY.RESEARCH PROTOCOL.: (PDF) Click here for additional data file. S2 FileBODY.DATA.: (XLS) Click here for additional data file. S1 TableBODY.CONSORT CHECKLIST FOR RANDOMISED CONTROL TRIAL.: (PDF) Click here for additional data file.

TITLE: Effect of risedronate on joint structure and symptoms of knee osteoarthritis: results of the BRISK randomized, controlled trial [ISRCTN01928173] To determine the efficacy and safety of risedronate in patients with knee osteoarthritis (OA), the British study of risedronate in structure and symptoms of knee OA (BRISK), a 1-year prospective, double-blind, placebo-controlled study, enrolled patients (40–80 years of age) with mild to moderate OA of the medial compartment of the knee. The primary aims were to detect differences in symptoms and function. Patients were randomized to once-daily risedronate (5 mg or 15 mg) or placebo. Radiographs were taken at baseline and 1 year for assessment of joint-space width using a standardized radiographic method with fluoroscopic positioning of the joint. Pain, function, and stiffness were assessed using the Western Ontario and McMaster Universities (WOMAC) OA index. The patient global assessment and use of walking aids were measured and bone and cartilage markers were assessed. The intention-to-treat population consisted of 284 patients. Those receiving risedronate at 15 mg showed improvement of the WOMAC index, particularly of physical function, significant improvement of the patient global assessment (P < 0.001), and decreased use of walking aids relative to patients receiving the placebo (P = 0.009). A trend towards attenuation of joint-space narrowing was observed in the group receiving 15 mg risedronate. Eight percent (n = 7) of patients receiving placebo and 4% (n = 4) of patients receiving 5 mg risedronate exhibited detectable progression of disease (joint-space width ≥ 25% or ≥ 0.75 mm) versus 1% (n = 1) of patients receiving 15 mg risedronate (P = 0.067). Risedronate (15 mg) significantly reduced markers of cartilage degradation and bone resorption. Both doses of risedronate were well tolerated. In this study, clear trends towards improvement were observed in both joint structure and symptoms in patients with primary knee OA treated with risedronate. BODY.INTRODUCTION: Osteoarthritis (OA) is a chronic, progressive disease that particularly affects weight-bearing joints such as hips and knees. The entire joint is affected by a complex combination of degradative and reparative processes, which alter the anatomy and function of articular cartilage, subchondral bone, and other joint tissues. Of the joints affected, knee OA in particular is a major cause of morbidity, often resulting in knee replacement [1-3]. Moreover, costs associated with OA are high – in the USA alone in 1991, the annual cost of knee replacements was estimated to be more than one billion dollars [4]. OA is normally the result of an interplay between systemic (e.g. age, obesity) and local (e.g. sports injury) factors that affect the joints of the body. Radiographic evidence has shown that joint-space narrowing (a surrogate marker for articular cartilage [5]), sclerosis of the subchondral bone, and the presence of osteophytes are typical structural features of OA. Current therapies for OA are largely aimed at providing symptom relief, and include a wide range of analgesics (e.g. nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 agents). In contrast, only limited data are available about therapies that modify the course of the disease or affect joint structure. Historically, OA has been considered a disease of the cartilage, but more recent evidence suggests that subchrondral bone is also involved in the pathogenesis, in both disease initiation and progression. For example, increased local bone turnover, decreased bone mineral content and stiffness, and decreased trabecular numbers have been observed in OA subchondral bone structure compared with normal bone [6-8]. There is an increased level of interest in subchondral bone as a therapeutic OA target, and, in particular, the possibility that drugs affecting bone metabolism might alter the progression of knee and hip OA. The Duncan-Hartley guinea pig model is a widely used spontaneous model of OA progression [9]. Several recent OA studies have evaluated this model for the effects of the antiresorptive agents bisphosphonates. For example, a comparative analysis of multiple bisphosphonates showed that only the group of nitrogen-containing bisphosphonates with pyridinyl sidechains demonstrated significant effects on the cartilage, although not all of these proved effective [10]. In a separate study using the guinea pig OA model, the pyridinyl bisphosphonate risedronate was shown to slow disease progression, as measured by the size and severity of cartilage lesions and the size of osteophytes, by up to 40% [11]. Based upon these preclinical studies, a clinical trial was performed in order to evaluate the effects of risedronate in patients with mild to moderate knee OA. The primary end points were changes in symptoms and function, with secondary end points of changes in joint structure or in markers of joint structure. BODY.MATERIALS AND METHODS: BODY.STUDY DESIGN AND SELECTION OF PATIENTS: The British Study of Risedronate in Structure and Symptoms of Knee OA (BRISK) was a 1-year prospective, double-blind, placebo-controlled study conducted in 10 centres in the UK. Male and female subjects aged 40 to 80 years with mild to moderate medial-compartment knee OA, diagnosed according to the clinical and radiological criteria of the American College of Rheumatology [12], were recruited into the trial. OA in at least one knee, designated the signal knee, was required to meet the following clinical and radiographic inclusion criteria. Clinical inclusion criteria were the presence of daily knee pain for at least 1 month out of the 3 months preceding the study, with at least one of the following: age >50 years, morning knee stiffness of <30 minutes, or knee crepitus. The radiographic criteria for inclusion were a joint-space width (JSW) of 2 to 4 mm in the medial tibiofemoral compartment in the semiflexed anterior–posterior (AP) view of the signal knee and a narrower width than in the lateral compartment of the same knee. Patients were also required to have at least one osteophyte in either the medial or the lateral compartment of the tibiofemoral joint. Major exclusion criteria were the presence of rheumatic diseases that could be responsible for secondary OA; use of intra-articular hyaluronic acid in the signal knee; knee injury or diagnostic arthroscopy of the signal knee in the 6 months preceding enrollment; a history of knee surgery (including arthroscopy requiring an incision of internal joint components) in the signal knee at any time; intra-articular corticosteroids in the 3 months preceding enrollment; the presence of non-OA causes of knee pain in the signal knee (e.g. anserine bursitis, fibromyalgia, or osteonecrosis); and the use of bisphosphonates within the 12 months preceding enrollment. The subjects gave their written, informed consent before entering the study, which was conducted in accordance with the International Conference on Harmonization (ICH) guidelines for Good Clinical Practice (GCP) and was approved by the UK Multicentre Research Ethical Committee (MREC). BODY.TREATMENT ASSIGNMENT: The subjects were randomly assigned in a 1:1:1 ratio to one of three arms to receive risedronate at 5 mg or 15 mg or placebo once daily for 1 year. Before randomization, patients were stratified according to their current use of oestrogen or a selective oestrogen receptor modulator. The subjects were instructed to take their study medication with at least 120 mL of water, 30 minutes before breakfast, or, if the medication was taken later in the day, at least 2 hours before or after food intake and at least 30 minutes before bedtime. They were instructed to take their study medication while they were upright and not to lie down for at least 30 minutes afterwards. BODY.SYMPTOM OUTCOME MEASURES: The outcome instrument for evaluation of risedronate efficacy on symptoms of OA was the Western Ontario and McMaster Universities (WOMAC) OA index [13]. The visual analogue scale (VAS) of the index was used, in which patients assessed each question using a 100-mm scale, with a higher score representing greater symptom severity. The total index score for the signal knee corresponded to the weighted composite of the 24 question scores standardized to a 100-point scale. Scores were also determined for the subscales of pain (5 questions), stiffness (2 questions), and physical function (17 questions). Other symptom outcome measures included the patient global assessment (PGA) of disease, consumption of pain medication, and the use of walking aids. For the PGA, patients answered the following question using a VAS: "Considering all the ways your OA affects you, how have you been in the last 48 hours?" Patients marked values on a scale from 0 to 100 mm. A step-down reduction in the use of pain medication was effected 5 days before all symptom evaluations. Patients were provided with approximately 30 tablets each of paracetamol (500 mg) and diclofenac (50 mg) to be used as the only pain medication 3 to 5 days before the baseline assessment and at visits at months 3, 6, and 12 (the exit visit). No pain medications were to be used 2 days before the scheduled evaluation date or on the day itself, with the exception of low-dose acetylsalicylic acid (<350 mg/day) for cardiac protection. Rescue analgesia was permitted during the study except for the 2-day washout period before each visit. BODY.STRUCTURE OUTCOME MEASURES: The outcome measure for assessment of joint structural changes was the mean change from baseline values in minimum JSW of the medial compartment of the knee. Radiographs of the knee were taken at baseline and at 1 year using a standardized radiographic method with fluoroscopic positioning of the joint in a semiflexed position [14,15]. By the use of this technique, the anterior and posterior rims of the tibia were aligned (to within 1 mm) for reproducible positioning. Radiographs were subjected to extensive quality control at the radiographic facility before dispatch to the Central Analysis Center [15]. Radiographs were read centrally and their quality control was rechecked before computer software was used to obtain the radiographic magnification. This was determined from measurement of a metal ball placed at the head of the fibula at the time of radiography and was used to adjust the computerized measurement obtained of the minimum medial compartment JSW [15]. The test–retest standard deviation of the difference between radiographs taken 2 days apart for this technique was approximately 0.2 mm, based upon repeat measurements in 199 subjects [15]. A retrospective analysis was performed taking into account the precision of the instrument. Retrospectively, clinically meaningful disease progression was defined as joint-space narrowing of ≥ 0.75 mm or a ≥ 25% loss from baseline values. The ≥ 0.75-mm value is almost four times the 0.2-mm standard deviation observed for the x-ray method. BODY.STRUCTURE–SYMPTOM RELATION: The relation between knee OA symptoms and radiographic joint-space narrowing was assessed retrospectively; the mean change in symptom scores between baseline to month 12 of the total WOMAC score and pain and function subscales was compared with the magnitude of change in JSW over the study period. BODY.BONE AND CARTILAGE MARKERS: Early-morning fasting urine and serum samples were collected at baseline and at months 3, 6, and 12 for assessment of markers of bone and cartilage turnover. Bone resorption was assessed by measurement of urinary levels of the N-terminal crosslinking telopeptide of type I collagen (NTX-I, Osteomark; OrthoClinical Diagnostics, High Wycombe, Bucks, UK) [16]. Bone formation was assessed by measurement of bone-specific serum alkaline phosphatase (Ostase, Beckman-Coulter, San Diego, CA, USA) [17] and cartilage degradation was assessed by measurement of urinary levels of C-terminal crosslinking telopeptide of type II collagen (CTX-II, Cartilaps, Nordic Bioscience, Herlev, Denmark) [18]. The intra-assay and interassay coefficients of variation were lower than 10%. BODY.EVALUATION OF SAFETY: Patient-reported adverse events (AEs) were recorded throughout the study. AEs were categorized using the Coding Symbols for Thesaurus of Adverse Reaction Terms (COSTART®) coding dictionary. Clinical laboratory measurements for safety monitoring were made throughout the study. Serious AEs were defined as any that resulted in death; were life threatening; resulted in hospitalization; resulted in persistent or significant disability or incapacity; or were judged to be medically significant. Upper-GI AEs included the following symptoms and conditions: substernal chest pain; duodenitis; dyspepsia; dysphagia; oesophagitis; gastritis; bleeding gastritis; gastro-oesophageal reflux; oesophageal bleeding; GI bleeding; haematemesis; melena; abdominal pain; ulcers (duodenal, oesophageal, peptic, gastric); bleeding ulcers (duodenal, peptic, gastric); perforated ulcers (duodenal, peptic, gastric); perforated and bleeding ulcers (duodenal, peptic, gastric); and reactivated ulcers (duodenal, peptic, gastric). BODY.STATISTICAL ANALYSIS: To ensure 80% power to detect a 20% effect of risedronate treatment versus placebo with respect to pain modification (quantified according to the WOMAC pain subscale, assuming a standard deviation of 70 mm on a 0- to 500-mm scale), a 1-year dropout rate of 20%, and a type I error rate of 5% without adjustment for two comparisons with placebo control, the sample size requirement was 100 patients per treatment group. Analyses were undertaken on the intention-to-treat (ITT) population. This was defined as all randomized patients who received at least one dose of study medication. All statistical analyses were performed using a two-sided statistical test with a type-I-error rate of 0.05. Baseline characteristics were compared using Fisher's exact test for categorical variables and the Kruskal–Wallis test for continuous variables. Extended Mantel–Haenszel tests with pooled centres as strata were used for end points with categorical responses. Analysis of variance (ANOVA) methods were used. Symptom analyses were adjusted for baseline value (PGA < WOMAC total or subscale value, as appropriate), pooled study centres, baseline use of oestrogen or selective oestrogen receptor modulators, gender, age, body mass index, and baseline JSW. Mean JSW analyses were adjusted for pooled study centres, baseline use of oestrogen or selective oestrogen receptor modulators, gender, age, body mass index, and baseline JSW as covariates. Each risedronate group was compared with the placebo group. For walking aids, the percentages were compared with placebo using the Cochran–Mantel–Haenszel test after adjusting for pooled centres. Individual AEs and the proportion of clinically meaningful JSW progressors were analysed using Fisher's exact test. The WOMAC scores were calculated in accordance with the WOMAC User's Guide [19]. The total scores were composed of subscales weighted as follows: pain = 42%, stiffness = 21%, and function = 37% [19]. For each subscale, the reported response was the patient's average. If at least two pain items, both stiffness items, or more than four physical function items were omitted, or if the patient's response was unclear, the items were regarded as invalid and the relevant subscale was not included. BODY.RESULTS: BODY.PATIENTS: Two hundred and eighty-five patients were considered eligible for the study and were randomized to treatment. Of these, 284 received at least one dose of study medication and were included in the ITT population, and 231 (81%) completed the study (placebo, n = 80; risedronate at 5 mg, n = 80; risedronate at 15 mg, n = 71) (Fig. 1). The number of patients who completed the study and the reasons for withdrawal were similar across treatment groups. Table 1 shows the baseline characteristics for the ITT population. These were similar between treatment groups; the average age of the patients was 63.3 years. There were no significant differences in the use of concomitant analgesics between treatment groups. Patients' compliance during study treatment, based on pill counts, was ≥ 83% and was comparable in the three treatment groups. BODY.SYMPTOM OUTCOME MEASURES: There was an improvement from baseline values in the symptom outcome measure of total WOMAC scores (weighted and unweighted) (unweighted not shown) and the subscales for all treatment groups (Fig. 2). The group given risedronate at 15 mg showed a trend towards improvement from baseline values, although the differences were not statistically significant (P values from 0.10 to 0.33). Assessment of PGAs revealed a statistically significant improvement with risedronate at 15 mg compared with placebo at 1 year (-19.4 for risedronate at 15 mg versus -5.7 for placebo, P < 0.001) (Fig. 3). Although all treatment groups showed a significant improvement from baseline values at 3 months, the improvement in the group receiving risedronate at 15 mg continued to increase with time, whereas the level of improvement with placebo or risedronate at 5 mg did not show any further improvement after 6 months. Analysis of the use of walking aids during study treatment showed a statistically significant difference in the proportion of patients who used walking aids in patients treated with risedronate at 15 mg (7 patients, 4% reduction) compared with placebo (21 patients, 8% increase) (P = 0.009) at 12 months compared with the proportion of patients that had reported using a walking aid during the previous year. BODY.STRUCTURE OUTCOME MEASURES: Assessment of the mean change from baseline values in minimum JSW in the medial compartment of the tibiofemoral joint at 1 year showed that there was a trend for patients receiving risedronate at 5 mg (JSW -0.08 ± 0.44 mm) or 15 mg (JSW -0.06 ± 0.25 mm). The change was greater in patients receiving placebo (JSW -0.12 ± 0.42 mm) compared with baseline values. Overall, the difference between treatment groups in loss of JSW at 12 months was not statistically significant (P = 0.275). The loss in JSW from baseline values was statistically significant only in the placebo group (P < 0.05). In terms of detectable progression (i.e. loss of JSW ≥ 25% or ≥ 0.75 mm), the analysis of the distribution of change from baseline values in JSW at 1 year showed a greater presence of detectable progression in the placebo (8%) and risedronate (5 mg) (4%) (P = 0.36) groups than in the risedronate (15 mg) group (1%) (P = 0.067). The patients with JSW loss of >0.75 mm included none of the patients treated with risedronate at 15 mg and 6% of the patients treated with placebo (P = 0.060). Similarly, only 1% of the patients treated with risedronate at 15 mg but 7% of the patients treated with placebo had >25% loss of JSW (P = 0.12). BODY.STRUCTURE–SYMPTOM RELATION: Figure 4 shows the relation between structure and symptoms for this population of patients. The mean WOMAC total score and the scores on the pain and function subscales increased (i.e. symptom severity increased) with increasing loss of JSW. In the group of patients with any loss of JSW, the mean changes of the WOMAC total score and the scores on the pain and function subscales at 1 year were -5.9 mm, -4.6 mm, and -6.3 mm, respectively, indicating that these symptoms were not increasing overall. In contrast, for the subset of patients with a loss in JSW of 40% or more, the corresponding mean changes were +1.4 mm, +6.0 mm, and +2.3 mm, indicating increased symptom severity in these patients concurrent with narrowing of their knee-joint space. BODY.MARKERS OF BIOCHEMICAL TURNOVER: Risedronate treatment significantly reduced markers of cartilage degradation (Fig. 5) and bone resorption compared with placebo. At 1 year, treatment with risedronate at 15 mg significantly decreased mean urinary CTX-II values, by -22.8% ± 5.35; urinary NTX-I was reduced by -32.9% ± 4.92 relative to baseline values (P < 0.05). Dose-dependent effects were also observed with the 5-mg dose compared with placebo, but to a lesser magnitude. This finding is consistent with the known pharmacologic effect of risedronate on bone turnover. At 1 year, CTX-II and NTX-I values in the placebo group were significantly higher than those in the risedronate 15-mg group (14.5% ± 5.4 and 17.2% ± 4.9 higher, respectively). Significant decreases in bone alkaline phosphatase were observed in the risedronate groups compared with placebo. At 1 year, the mean decreases in the groups receiving risedronate at 15 mg and 5 mg were 29.1% ± 2.6 and 19.5% ± 2.5, respectively, compared with a mean decrease of 2.7% ± 2.5 in the placebo group (P < 0.001). BODY.SAFETY: The frequencies of AEs were similar in the two treatment groups (Table 2). There were no clinically meaningful differences between groups in the percentage of patients with AEs in any body system and there were no significant differences in routine clinical chemistry parameters between the risedronate groups and the placebo group. The numbers of patients who dropped out of the study because of AEs were similar. Overall, 34 patients reported a total of 53 serious AEs. Investigators considered four serious AEs as possibly related to study treatment; two of these (rash and diarrhea) were in patients treated with placebo and two (anaemia and increased general joint pain) were in patients treated with risedronate at 5 mg. Table 2 provides a summary of adverse events for the ITT population and the frequency of the overall GI AEs and the most common upper-GI AEs. Forty-seven upper-GI events were reported in 38 patients, of which abdominal pain and dyspepsia were the most frequently reported. The majority of the upper-GI AEs occurred in patients with a history of GI disease; there were no significant differences between the groups given risedronate and the group given placebo in the incidence of upper-GI AEs in these patients. BODY.DISCUSSION: Increased evidence of the role of bone in both the initiation and progression of OA has resulted in an interest in drugs that affect bone metabolism and might slow or even halt the process of joint degeneration [6]. The early findings reported here suggest that the bisphosphonate risedronate may have disease-modifying effects in patients with knee OA. A recent cross-sectional study also suggests an association between antiresorptive treatments (oestrogen or bisphosphonate) and improved symptoms and/or decreased bone marrow abnormalities [20]. Positive trends were observed with risedronate treatment with regard to symptomatic improvement, as assessed by the WOMAC index. Treatment with risedronate at 15 mg resulted in a consistent trend in improvement in WOMAC scores, whereas the group receiving placebo showed less improvement. The group receiving risedronate at 15 mg showed a significant improvement in the PGA of OA compared with placebo. Similarly, the percentage of patients who used a walking aid during the study decreased in the group treated with risedronate at 15 mg, contrasting with an increase in the placebo group. While the differences in JSW among the groups were not significant, there was a trend for less loss in the risedronate (15 mg) than in the placebo group. When the data were analysed in a post hoc manner to identify patients with detectable progression (i.e. approximately four times the precision of the measurement), 8% of patients receiving placebo and 1% of patients receiving 15 mg risedronate were found to exhibit this degree of progression. Additionally, risedronate significantly reduced levels of bone resorption and cartilage degradation, as assessed by NTX-I and CTX-II markers, respectively. Despite these encouraging results, they were not confirmed in multicentre studies of risedronate treatment for 2 years using a similar protocol [21]. The following provides perspectives comparing the two studies. In our study, there was a dose-dependent trend for improvement in the WOMAC score. In the 2-year North American study, which enrolled 1,232 patients with knee OA, the placebo effect was approximately twice that observed in our study, and was comparable to the magnitude of change observed in the group given 15 mg risedronate in our study. Both studies showed significant decreases in CTX-II, the marker of cartilage degradation, but these were not associated with an attenuation of joint-space loss This lack of association may be related to the length of observation in these studies. Reijman and colleagues [22] observed 1,235 men and women with OA, followed up over an average period of 6.6 years. The subjects with baseline CTX-II levels in the highest quartile had a sixfold risk of progression of knee OA, defined as a decrease in JSW ≥ 2 mm, in comparison with subjects in the lowest quartile [22]. This suggests that an enriched population of subjects with an elevated rate of cartilage loss combined with a longer study period may be required in order to observe significant treatment effects. Our study is one of the first to suggest a correlation between symptoms and structure in OA, although preliminary results with doxycycline in the treatment of obese women with knee OA have reported a significant reduction in the proportion of follow-up visits in which a clinically significant increase in pain occurred, favouring treatment over placebo, and coinciding with a decrease in joint-space narrowing [23]. This finding is important, because it runs contrary to previous results, which have suggested a poor correlation between these disease features [24,25]. The limitations of our study include the small number of patients (n 26,27]. The results highlighted the importance of medial tibial plateau alignment with the central x-ray beam and showed that the standard clinical view of a standing extended knee is subject to considerable variability. In contrast, the fluoroscopic technique is well validated and is less variable in test-retest performance [15,28]. Further studies are required to further explore the possible correlation between symptoms and structure observed in our study. If validated, this relation may allow physicians to use the assessment of pain, perhaps in combination with a biomarker such as CTX-II, as a surrogate for other measures of disease progression BODY.CONCLUSION: This study is one of the first to show a correlation between symptoms and joint structure changes in knee OA. While our findings were suggestive of a beneficial effect of risedronate treatment on preservation of bone and cartilage, these trends seen in this study have not been observed in larger, multicountry cohorts. BODY.ABBREVIATIONS: AE = adverse event; BRISK = British Study of Risedronate in Structure and Symptoms of Knee OA; CTX-II = C-terminal crosslinking telopeptide of type II collagen; GI = gastrointestinal; ITT = intention-to-treat; JSW = joint-space width; NTX-I = N-terminal crosslinking telopeptide of type I collagen; OA = osteoarthritis; PGA = patient global assessment; VAS = visual analogue scale; WOMAC = Western Ontario and McMaster Universities [OA index]. BODY.COMPETING INTERESTS: This manuscript was sponsored by a grant from Procter & Gamble. BODY.AUTHORS' CONTRIBUTIONS: TDS, JFB, DJV, and JMM planned the study and prepared the manuscript. GAC performed the statistical analysis. JCB-W supervised radiological measurements. PG performed the marker assays. PGC assisted with the manuscript and recruited patients. All authors read and approved the final manuscript.

TITLE: A double-blind, randomized, placebo-controlled pilot trial to determine the efficacy and safety of ibudilast, a potential glial attenuator, in chronic migraine ABSTRACT.BACKGROUND: Chronic migraine (CM) is problematic, and there are few effective treatments. Recently, it has been hypothesized that glial activation may be a contributor to migraine; therefore, this study investigated whether the potential glial inhibitor, ibudilast, could attenuate CM. ABSTRACT.METHODS: The study was of double-blind, randomized, placebo-controlled, two-period crossover design. Participants were randomized to receive either ibudilast (40 mg twice daily) or placebo treatment for 8 weeks. Subsequently, the participants underwent a 4-week washout period followed by a second 8-week treatment block with the alternative treatment. CM participants completed a headache diary 4 weeks before randomization throughout both treatment periods and 4 weeks after treatment. Questionnaires assessing quality of life and cutaneous allodynia were collected on eight occasions throughout the study. ABSTRACT.RESULTS: A total of 33 participants were randomized, and 14 participants completed the study. Ibudilast was generally well tolerated with mild, transient adverse events, principally nausea. Eight weeks of ibudilast treatment did not reduce the frequency of moderate to severe headache or of secondary outcome measures such as headache index, intake of symptomatic medications, quality of life or change in cutaneous allodynia. ABSTRACT.CONCLUSION: Using the current regimen, ibudilast does not improve migraine with CM participants. BODY.INTRODUCTION: Chronic migraine (CM) is a disabling and undertreated disorder1 which represents a clinically challenging problem that impacts significantly on the quality of life of affected individuals and imposes a large economic burden upon society due to lost productivity.2,3 Despite this burden, the pathophysiology behind migraine and especially the processes of migraine chronification remain poorly understood. A growing body of preclinical and clinical evidence suggests that acquired sensitization plays a significant role in the pathology of migraine and the process of chronification.4 Recent evidence supports the importance of neuronal–glial interactions as a potential mechanism for chronic pain. Glia are located in the central nervous system, and under basal condition, they perform housekeeping functions and maintain neural homeostasis.5 However, when glia are activated, they become immunoresponsive and modulate pain by releasing neuroexcitatory signals that can increase neuronal excitability,6,7 activate neighboring glia, and produce nociceptive mediators (such as nitric oxide, excitatory amino acids, and proinflammatory cytokines).8 Nonclinical models support a role of neuron–glia interactions in the initiation of migraine attacks, as glia have been shown to release a range of inflammatory cytokines and chemokines in animal models evoking some features of migraine.9 In nonclinical models, blockade of glial activation and proinflammatory mediators released by glia caused reduction in hypersensitivity and allodynia, respectively.10,11 Additionally, a clinical study has reported elevated levels of proinflammatory cytokines in the cerebrospinal fluid of CM participants.12 Such tonically increased glial activity may underlie the progression from episodic to CM.13 Furthermore, in a separate study, levels of the chemokine interleukin 8 were found to be elevated in the jugular venous blood of patients during a migraine attack.14 Interleukin 8 can be synthesized by glia in response to other proinflammatory mediators including tumor necrosis factor α, lending additional support to the neurogenic inflammation model of migraine.15 Thus, it seems likely that nociceptive signaling during migraine attacks releases mediators that activate glia. Activated glia then release a range of proinflammatory mediators such as cytokines, chemokines, reactive oxygen species, and prostaglandins, which not only facilitate pain transmission but also activate further glia, creating a positive feedback loop. After a stimulus is resolved, experimental evidence suggests that microglia may remain “primed,” entering a state in which they do not actively produce proinflammatory substances, yet they over-respond to subsequent stimuli, resulting in increased proinflammatory cytokine release and an exaggerated pain response.16 This could lead to an increase in migraine frequency as triggers that were previously subthreshold may now initiate headache. Although glial involvement in the pathogenesis of migraine and in the transformation of episodic migraine to CM has been suggested by a number of authors,9,13,17–19 to our knowledge no treatments targeting this mechanism have been trialed specifically in high frequency migraineurs. However, encouraging results from an open-label retrospective study in participants with varying chronic daily headache phenotypes following treatment with the potential glial attenuator minocycline have been reported.20 This study, published only in abstract form, found a significant decrease in headache frequency following 2 months of minocycline administration when used as an adjunctive preventative medication.20 However, the efficacy of minocycline in animal studies, suggests that it is only effective before chronic pain is established and not subsequently,10 which may not be applicable to chronic pain patients. Ibudilast is a nonselective phosphodiesterase inhibitor that has been used in Japan for 20 years to treat bronchial asthma,21 post-stroke dizziness,22 and ocular allergies.23 Ibudilast inhibits tracheal smooth muscle contractility24 and platelet aggregation,25 improves cerebral blood flow,22 and attenuates allergic reactions.26,27 As ibudilast is administered orally, is well tolerated, and has good blood–brain barrier permeability,28–30 it is a suitable candidate for the treatment of chronic pain. In nonclinical models, ibudilast attenuated microglial activation (reduced withdrawal behaviors), reduced production of proinflammatory cytokines, enhanced the production of anti-inflammatory cytokines, and improved the safety profile of opioids (reduced tolerance and attenuated reward and dependence).23,31–33 Moreover, ibudilast exhibited immunomodulatory actions in multiple sclerosis patients30 and also displayed inhibition of proinflammatory cytokines from peripheral leukocytes.34 In the present study, the efficacy of the putative glial attenuator ibudilast in participants with high frequency migraine was investigated. The study investigated the efficacy of ibudilast on the frequency of moderate to severe headache and other headache indices (such as headache index, headache-related questionnaires, and acute symptomatic medication intake) in CM patients over 8 weeks of ibudilast treatment compared with placebo treatment. BODY.METHODS: BODY.STUDY APPROVAL: Ethical approval was obtained from the Human Research Ethics Committee of the Royal Adelaide Hospital, Adelaide, South Australia. All the participants gave their written informed consent before the commencement of the study. The study was conducted at the Pain and Anaesthesia Research Clinic, Royal Adelaide Hospital, Adelaide, Australia, and in accordance with the Principles of International Conference on Harmonization Good Clinical Practice, the Declaration of Helsinki, and the Australian National Statement on Ethical Conduct in Research Involving Humans. The study was of double-blind, randomized, placebo-controlled, two-period crossover design. Participants were randomized to initially receive either ibudilast or placebo treatment for 8 weeks. Subsequently, the participants underwent a 4-week washout period followed by a second 8-week treatment block with the alternative treatment (Clinicaltrials.gov NCT01389193). BODY.PARTICIPANT SELECTION: Participants were recruited from June 2013 to May 2015. Criteria for CM were defined by the revised second edition of the International Classification of Headache Disorders (ICHD-II).35 Key inclusion criteria were: Men and women aged between 18 and 65 years Migraine with or without aura, as diagnosed according to the ICHD-II36 Onset of migraine before 50 years of age Headache for ≥15 days per month Migraine-like headache for ≥8 days per month, as per the International Headache Society (IHS) guidelines37 BODY.KEY EXCLUSION CRITERIA: Participants were excluded from the study on the basis of the following criteria: change in type or dose of migraine prophylactic medication in last 3 months, medication overuse headache as diagnosed according to the ICHD-II35, posttraumatic headache as diagnosed according to the ICHD-II36, another dominant chronic pain condition, an active inflammatory disease such as rheumatoid arthritis, a history of recent cerebrovascular disorder, inability to provide written informed consent, inability to read and write in English, presence of severe psychological/psychiatric disorders, recent history of significant trauma, recent history of drug or alcohol abuse, clinically significant findings on screening blood sample results, current malignancy, known hypersensitivity to ibudilast or excipients in Ketas® formulation, renal or hepatic impairment, pregnancy, lack of adequate contraception, and breastfeeding. BODY.STUDY MEDICATION: The study used a delayed-release ibudilast product, Ketas®, which was obtained from Kyorin Pharmaceuticals Industries, Ltd (Tokyo, Japan). To blind the treatment allocation, the Ketas® capsule was overencapsulated with the space filled with microcrystalline cellulose. The placebo capsule was of the same size and color and filled with microcrystalline cellulose, so that the active and placebo medications were identical in appearance and weight. Each active capsule contained 10 mg ibudilast. Four capsules were self-administered by participants orally, twice daily for each 8-week treatment block. No treatment was administered during the washout. BODY.STUDY SCHEDULE: BODY.4 WEEKS PRIOR TO BASELINE VISIT: At screening, potential participants underwent, Medical history including medication history Headache diagnosis based upon retrospective information from the participant confirmed using baseline headache diary data Completion of the Hospital Anxiety and Depression Scale Physical examination Urine pregnancy test for women of childbearing potential Urine drug screen to rule out the presence of nonprescribed drugs of abuse Blood samples to assess hematology (4 mL), biochemistry including renal function and hepatic function (8 mL). After successful completion of the screening visit and confirmation of study eligibility, participants were provided with a headache diary to complete at least 4 weeks prior to the baseline visit to confirm study eligibility. Standardized education and instructions on how to complete the diary was provided by the experimenter, and the participants were required to fill in the headache diary daily. The headache diary recorded a comprehensive assessment of the headache experienced by participants and consisted of head pain characteristics, headache frequency, average headache intensity (11-point numerical rating scale), duration of headache (number of hours), and intake of symptomatic headache treatments (timing, type, and amount of medication consumed). The headache index was calculated by the summation of headache duration (hours) with headache intensity (11-point numerical rating scale). The dose of opioid-containing analgesics used by each patient was converted to oral morphine equivalent dose based on the local Royal Adelaide Hospital Pain Management Unit opioid conversion table. BODY.BASELINE VISIT: During the baseline visit, the headache diary was collected, and participants who were eligible to continue in the study were randomized to either ibudilast or placebo treatment in the first period. Participants were asked to complete two brief self-administered questionnaires. The 6-point Headache Impact Test (HIT-6) evaluated the impact of headache on the quality of life, reflecting upon the previous month,38 and the 12-point Allodynia Symptom Checklist (ASC-12) indicated whether, and quantified how, increased pain sensitivity impacted upon activities of day-to-day life.39 About 14 mL of blood was collected to assess safety blood profile (blood biochemistry [including the assessment of renal function], liver function tests and hematology). Participants were advised to take only the minimum amount of medications required to control their pain; however, they were not specifically informed to reduce their analgesic intake. Participants were asked to complete another headache diary for the 8-week treatment period. The study medication was dispensed to the participants prior to discharge. Participants were required to complete the headache diary throughout the two treatment periods, during the washout phase and for 4 weeks after ceasing the study medication. BODY.IN STUDY VISITS: Inspection of headache diary, assessment of adverse events, completion of questionnaires, and safety bloods were taken at weeks 2, 4, and 8 of each period. Figure 1 shows the flowchart of the study design. BODY.STATISTICAL ANALYSIS: All the participants who received at least one dose of the study medication were included in the safety population. However, the efficacy data were only analyzed for completers, given the crossover nature of the trial. Graphpad Prism 6.0 (GraphPad Software; San Diego, CA, USA) was used for all statistical analysis. The Wilcoxon matched pairs signed rank test was used to calculate the study medication adherence between the CM participants while in different treatment groups. Two-way analysis of variance tests with repeated measures and with Sidak adjustments were used to determine the differences between treatments in multiple comparisons were used to calculate frequency of moderate to severe headache experienced, headache index, frequency of symptomatic medication intake, average opioid intake, HIT-6 scores, and ASC-12 scores. Missing values were replaced by the last observed value for that variable (last observation carried forward). All results are presented as mean ± standard deviation (SD) unless stated otherwise. Significance was set at P<0.05. BODY.RESULTS: BODY.DEMOGRAPHICS OF CM PARTICIPANTS: A total of 33 patients were randomized to treatment. Fourteen CM participants completed both the treatments of the study and returned completed headache diaries suitable for analysis. Four CM participants completed both the treatments of the study but did not return adequate diaries for analysis and therefore were excluded from the analysis (Figure 2). Fifteen participants (nine received ibudilast) did not complete the study and hence were also excluded from the analysis. Of these fifteen participants, two participants lost interest in the study; one was excluded due to work commitment; four were excluded because of loss to follow-up; one was excluded as that participant had changed his medication; one was excluded after receiving 1 week of treatment as she was found to be pregnant (she was on placebo); one was excluded after experiencing heart palpitation; four participants were withdrawn by the investigator because of worsening headaches; and one participant was excluded by the investigator as the patient experienced tingling sensation. Demographics of the participants are listed in Table 1. The mean age was 46 years (SD 13). The study consisted of 11 men and 22 women. The average duration of headache was 21 years (SD 15). The mean daily morphine equivalent dose was 4.2 mg (SD10). BODY.COMPLIANCE TO STUDY MEDICATION AND SAFETY: In this study, all the participants had good adherence to study medications as demonstrated with the return of unused study capsules. There was no treatment group difference (P=0.6) with the number of capsules returned (placebo vs ibudilast [mean {SD}: 2.4 {2.4} vs 7.7 {15.6}]). Adverse events experienced in the study were of mild intensity, and all the reported adverse events are listed in Table 2. Seventeen of the twenty-three (60%) CM participants who were on ibudilast treatments reported adverse events, whereas eight of the twenty-three (35%) CM participants reported adverse events during placebo treatments. The most frequent adverse event in both the groups was worsening migraine, reported by 4/28 under ibudilast and 2/23 on placebo. The next most frequently reported adverse event was nausea, reported by 4/28 under ibudilast and by no participant on placebo. BODY.HEADACHES: When CM participants were on ibudilast treatment, they did not experience reduced frequency of moderate to severe headache compared with placebo treatment (P=0.9) in either treatment weeks 1–4 (mean difference −0.9, 95% confidence interval [CI] −9.1 to 7.3) or treatment weeks 5–8 (mean difference 0.1, 95% CI −8.1 to 8.3). However, irrespective of treatments, in treatment weeks 5–8 of each block, there was a significant reduction (P=0.02) in the frequency of moderate to severe headaches (Figure 3). Likewise, when CM participants were on ibudilast treatment, they did not experience a reduction in headache index when compared with placebo treatment (P=0.9) in either treatment weeks 1–4 (mean difference −5.7, 95% CI −56.6 to 45.2) or treatment weeks 5–8 (mean difference −0.4, 95% CI −51.3 to 50.5). However, in treatment weeks 5–8, there was a significant reduction in the headache index (P=0.003) (Figure 4). BODY.ACUTE SYMPTOMATIC MEDICATION INTAKE AND SELF-REPORTED QUESTIONNAIRES: No treatment differences were detected in the frequency intake of symptomatic medication (P=0.6) in either treatment weeks 1–4 (mean difference −2.6, 95% CI −11.7 to 6.6) or treatment weeks 5–8 (mean difference −1.9, 95% CI −11 to 7.3) (Figure 5A). The oral morphine equivalent was also calculated for the medication that was taken by the CM participants; however, no treatment differences were detected (P=1) in either treatment weeks 1–4 (mean difference −0.5, 95% CI −11.2 to 10.2) or treatment weeks 5–8 (mean difference 0.5, 95% CI −10.2 to 11.2) (Figure 5B). Scores on the HIT-6 and ASC-12 questionnaires did not differ between the treatment groups during any study time period (baseline, weeks 2, 4, and 8 of treatment); P=0.3 and P=0.9, respectively (data not shown). BODY.DISCUSSION: Ibudilast caused no change in the frequency of moderate to severe headache when compared with placebo treatment. Furthermore, other headache indices such as headache index, frequency of acute symptomatic medication intake, HIT-6 scores or ASC-12 scores were not reduced when CM patients were on ibudilast treatment. Additionally, worsening migraine was reported at a higher frequency under ibudilast treatment rather than on placebo. However, given the small sample sizes used in this study, lack of statistical significance should be considered with caution, but there was no trend for a benefit with ibudilast. Interestingly, during treatment weeks 5–8 of each period, the frequency of moderate to severe headache and headache index for both the treatment groups were significantly reduced compared to treatment weeks 1–4. This observation could be an indicator of participants having a positive expectancy of the study drug.40 Even though ibudilast may not be an effective treatment for CM, this study further provides a safety profile of ibudilast in a homogeneous migraine cohort. The adverse events experienced by participants were mild and transient in nature, and most of the adverse events were resolved once ibudilast was halted. Our results are in agreement with a recently published finding by our group, which found ibudilast to have no effect in reducing headache index or in reducing opioid intake in medi cation overuse headache patients.41 However, the study also measured toll-like receptor (TLR) reactivity from peripheral blood mononuclear cells and found ibudilast to significantly reduce the proinflammatory cytokine (interleukin 1β) levels from ex vivo stimulation with TLR-2 or TLR-4 agonists. This finding confirmed that ibudilast was likely to be inhibiting an inflammatory pathway, at least peripherally. However, the peripheral target may be different to the mechanism that is responsible for the headache or the treatment period may have been insufficient to reverse the long-term changes associated with CM or may not have been acting centrally. It is unclear from this study whether glia are involved in CM. Ibudilast has demonstrated good central nervous system partitioning28 but as it was not found to have any effect in the clinical endpoints, it suggests that the mechanisms responsible for CM may be signaling through another pathway that is currently unidentified, or neurons may play a stronger role. Thus more research would be required to address whether glial activation is a likely contributor for CM. The recent demonstration of imaging of activated glia in patients with chronic low back pain provides a potential biomarker for future studies.42 Even though there have been reports of pro- and anti-inflammatory cytokines being a predictor of headaches, the examination of peripheral targets may not be adequate. In a CM study, elevation of tumor necrosis factor α was found in the cerebrospinal fluid but not in the plasma,12 indicating that peripheral biomarkers might not be helpful. BODY.LIMITATIONS: The sample size for this study was small as the required number of participants could not be recruited within the time frame, so the study ended prematurely (intended target was 40 CM participants). This was due to the difficulty in finding CM participants who fitted the criteria, and this is common in research involving chronic headache patients.43,44 BODY.CONCLUSION: The present study showed ibudilast dosed at 40 mg twice daily for 8 weeks is not a promising treatment for CM. However, this study adds to the literature of the safety and tolerability of ibudilast in a CM cohort, which to our knowledge has not been investigated previously.

TITLE: AIO LQ-0110: a randomized phase II trial comparing oral doxycycline versus local administration of erythromycin as preemptive treatment strategies of panitumumab-mediated skin toxicity in patients with metastatic colorectal cancer ABSTRACT.BACKGROUND: Dermatologic toxicities, especially akne-like skin rash, are the most common side-effects associated with anti-epidermal growth factor receptor (EGFR) therapy. Preemptive treatment with oral tetracyclines is recommended as a standard. Topical prophylactic options have thus far not been compared to tetracyclines. In the current study, we sought to establish an alternative topical treatment. ABSTRACT.PATIENTS AND METHODS: In this multicentre, randomized, open-label phase II study patients with (K)Ras-wildtype colorectal cancer receiving panitumumab were randomized (1:1) to receive either doxycycline 100 mg b.i.d. (standard arm) or erythromycin ointment 2% followed by doxycycline in case of insufficient activity. The primary endpoint was the percentage of patients developing no skin toxicity ≥ grade 2 at any time during the first 8 weeks of panitumumab treatment. Skin toxicity was assessed using the NCI CTCAE v 4.0. Secondary endpoints comprised the assessment of skin toxicity using a more thorough grading system (WoMo score), evaluation of skin-related (DLQI) and global quality of life (EORTC QLQ C30). ABSTRACT.RESULTS: In total, 88 patients were included in this trial. 69% of the patients in the erythromycin arm suffered from skin toxicity of grade ≥ 2 versus 63% in the standard arm (P = n.s.). However, as per WoMo score significantly more patients in the erythromycin arm developed moderate or severe skin toxicity at earlier time points. Skin related and overall quality of life was comparable between both arms. ABSTRACT.CONCLUSIONS: Based on this data erythromycin cannot be regarded as an alternative to doxycycline as prevention of EGFR-related skin toxicity. BODY.INTRODUCTION: Dermatologic toxicities are the most common side-effects associated with anti-EGFR therapy and they are experienced by the majority of patients undergoing EGFR inhibitor therapy. While toxicities are mild (grade 0-1) to moderate (grade 2) in most patients, about 15-20% exhibit higher grade toxicities leading to medical and psychosocial effects that may result in poor patients’ compliance with lower adherence to cancer therapy, more dose delays, and interruption or discontinuation of antineoplastic therapy [1, 2]. Finally, dermatologic toxicities may contribute to a significantly reduced quality of life [2, 3, 4]. Acneiform rash, usually developing within the first 2 to 4 weeks of therapy, is by far the most frequent type of skin toxicity caused by EGFR antibody treatment. Management guidelines for the prophylaxis and treatment of EGFR inhibitor-mediated dermatologic toxicities usually recommend prophylactic use of tetracyclines and special skin care. Indeed, these recommendations are based on few randomized trials: Jatoi et al. randomized 62 patients treated with anti-EGFR antibodies or tyrosine kinase inhibitors (TKIs) between tetracycline and placebo. While the overall incidence of skin toxicity was not reduced, a significant decrease of severity was observed. Of note, patients treated with tetracycline reported improved skin-related quality of life as assessed by the SKINDEX-16 quality of life (QoL) questionnaire [5]. In a subsequent similar study including 65 patients Jatoi and coworkers could not confirm these findings [6]. Scope et al. randomized 48 colorectal cancer patients treated with cetuximab between minocycline and placebo. Minocycline significantly decreased moderate to severe itch. Likewise, a trend toward a decreased incidence of moderate to severe rash was noticed (20% vs. 42%, P =.13) [7]. Deplanque et al. randomized 147 patients with non-small-cell lung cancer (NSCLC) between treatment with erlotinib alone or in combination with doxycycline. Doxycycline did not reduce the incidence of folliculitis over untreated patients (71% vs. 81%, P =.117) but significantly reduced its severity (P =.001) [8]. Melosky et al. randomized 150 patients with NSCLC treated with erlotinib between prophylactic minocycline, reactive minocycline and no treatment. The incidence of skin toxicity was 84% regardless of treatment arm. Prophylactic treatment with minocycline, however, significantly lengthened the time to the most severe grade of rash. Grade 3 rash was significantly higher in the no-treatment arm [9]. Arrieta et al. randomized 90 patients with NSCLC treated with afatinib between preemptive tetracycline and only reactive treatment. Tetracycline reduced the rash incidence of any grade (44.5% vs. 75.6%, P =.046) and the severity of grade ≥ 2 (15.6% vs. 35.6%, P =.030) over reactive treatment [10]. Finally, Lacouture et al. randomized 114 patients with NSCLC treated with dacomitinib between preemptive doxycycline and placebo. Doxycycline significantly reduced the incidence of select dermatologic adverse events of interest (SDAEI) grade ≥ 2 by 50% (P =.016) [11]. In all, it appears to be justified to recommend a prophylaxis with tetracyclines such as doxycycline or minocycline. Two randomized studies confirmed that tetracyclines should be used as prophylaxis and not as an early intervention: Firstly, the so-called STEPP trial randomized 95 patients with metastatic colorectal cancer (mCRC) to be treated with panitumumab in combination with chemotherapy to either preemptive treatment for skin toxicity including doxycycline 100 mg b.i.d. or to receive the same regimen reactively. Incidence of skin toxicity grade ≥ 2 was significantly reduced by preemptive treatment compared with reactive treatment (29% vs. 62%). Moreover, patients randomized to preemptive treatment reported improved QoL according to Dermatology Life Quality Index (DLQI) especially between week 2 and 3 [3]. Secondly, the so-called J-STEPP trial confirmed these data using a comparable study design in a Japanese population with mCRC. Comparable to the STEPP trial, the incidence of ≥ grade 2 skin reactions was decreased with preemptive treatment compared with reactive treatment (21.3% vs. 62.5%, P <.001) [12]. Thus far, no data comparing oral antibiotics with a local treatment have been reported. In order to avoid a long term exposure to oral antibiotics a preemptive topical skin treatment could be useful. Moreover, topical agents are more convenient than oral antibiotics, and compliance might be better. In the present trial we sought to compare a local treatment using erythromycin followed by doxycycline in the case of insufficient activity with doxycycline. Erythromycin seemed to be very suitable as it belongs to standard treatment for acne vulgaris [13]. BODY.RESULTS: BODY.PATIENTS: In total, 88 patients were randomized by 11 sites between July 2011 and October 2014. 80 out of 88 patients were evaluated (41 patients in the doxycycline arm, 39 patients in the erythromycin arm). Reasons for exclusion were: withdrawal of consent, no therapy started (n = 4), no post-baseline skin toxicity assessment (n = 1); treatment not started due to RAS mutation (n = 2), investigator´s decision (n = 1). Patients´ and major tumor-related baseline characteristics are depicted in Table 1. Table 1BODY.MAJOR PATIENTS´ AND TUMOUR CHARACTERISTICS: ParameterDoxycyclineErythromycinTotalAgeN 40* 39 79  Mean ± SD (years) 68.1 ± 12.4 69.1 ± 9.4 68.6 ± 11  Median (years) 69.5 70.3 70.2 Gender N 41 39 80  Female 13 (32%) 18 (46%) 31 (39%)  Male 28 (68%) 21 (54%) 49 (61%) Cancer location N 41 38* 79  Colon 23 (56%) 18 (47%) 41 (52%)  Rectum 18 (44%) 20 (53%) 38 (48%) Performance status N 39* 38* 77  ECOG 0 16 (41%) 17 (45%) 33 (43%)  ECOG 1 19 (49%) 19 (50%) 38 (49%)  ECOG 2 4 (10%) 2 (5%) 6 (8%) *Data missing. BODY.PRIMARY END POINT: The percentage of patients developing skin toxicity according to NCI CTC criteria is shown in Table 2. In the erythromycin arm, 69% suffered from skin toxicity grade ≥ 2 (95% confidence interval [CI]: 52% to 83%; primary endpoint). In the standard arm with doxycycline the toxicity rate with grade ≥ 2 amounted to 63% (95% CI: 47 – 78%). Thus, the primary endpoint was not met. Table 2BODY.SKIN TOXICITY GRADED ACCORDING TO NCI CTCAE 4.0, WORST PER PATIENT: NCI CTCAE gradeDoxycyclineErythromycinTotalN 41 39 80 0 / no toxicity 3 (7%) 3 (8%) 6 (8%) 1 12 (29%) 9 (23%) 21 (26%) 2 18 (44%) 12 (31%) 30 (38%) 3 8 (20%) 15 (38%) 23 (29%) P =.27 (Mantel-Haenszel test for trend, exact version). Although this randomized phase II study was not powered for formal statistical comparisons of the two treatment groups, results from additional statistical calculations are provided in a descriptive way: Using Fisher's exact test no significant differences between both arms were seen (P =.64). The odds ratio, with values > 1.0 indicating higher toxicity, was 1.29 (95% confidence interval: 0.46 – 3.67; one-sided 95% confidence limit for inferiority of the erythromycin arm: 3.15). In absolute values, the rate difference of 6% more toxicity in the erythromycin group has a 95% confidence interval (according to HAUCK-ANDERSON) ranging from −16% to + 28%. Figure 1 shows the time to the onset of skin toxicity of NCI CTCAE grade ≥ 2. In the doxycycline arm median time was 43 days (day one of cycle 4), in the erythromycin arm median time was 29 days (day one of cycle 3) without statistical significance in the corresponding logrank test (P =.68). Figure 1BODY.TIME TO SKIN TOXICITY NCI CTCAE GRADE ≥ 2 (FULL ANALYSIS SET [N = 80]): BODY.SKIN TOXICITY GRADED ACCORDING TO WOMO SCORE: The total score result from the WoMo assessment over time (individual cycles) show a constantly higher score in the erythromycin group (data not shown). When the maximum WoMo score by patient over time is analyzed (Table 3), the difference amounts to almost 10 points which is about half of the standard deviation. Table 3BODY.SKIN TOXICITY ACCORDING TO WOMO SCORE: WoMo scoreDoxycyclineErythromycinTotalN 41 39 80 Mean ± SD 25.6 ± 16.7 34.1 ± 19.8 29.7 ± 18.7 Median 23.1 32.1 29.5 Quartiles 14.5 - 34.8 21.9 - 47.2 16.1 - 40.9 Range 0 - 68.2 0 - 88.1 0 - 88.1 A significantly higher number of patients treated in the erythromycin arm developed moderate or severe skin toxicity according to WoMo score, i.e. ≥ 20 points (30/39 versus 23/41 patients, P = .049); moreover, a higher number of patients had severe skin toxicity (WoMo score ≥ 40 points 14/39 versus 8/41 patients; P =.101). Analyzes based on worsening of the WoMo score were performed. It took one cycle longer to appearance of a moderate to severe skin toxicity (WoMo score ≥ 20) with doxycycline compared to erythromycin. Figure 2 shows the time to increase of the WoMo score to ≥ 20. An obvious difference between both arms appeared on day 15 (day one of cycle 2) and this remained discernible thereafter indicating a clear trend towards superior efficacy of the standard arm using doxycycline (P =.069). Figure 2BODY.TIME TO WOMO SCORE ≥ 20 (FULL ANALYSIS SET [N = 80]): BODY.QUALITY OF LIFE: The mean values of the EORTC QLQ C30 global health score are balanced at the beginning of the first treatment cycle with about 52 points for each group (albeit based on a lower number of valid questionnaires in the erythromycin group; Table 4). Over time, the mean values amount to 59 in the erythromycin arm and decline to 47 points in the doxycycline arm (at the beginning of cycle 4). The between-group differences range between 5 and 10 points per cycle. Table 4BODY.QUALITY OF LIFE GRADED ACCORDING TO EORTC QLQ C30: GLOBAL HEALTH SCORE: Erythromycin arm Cycle 1 2 3 4 EOT* N 32 36 27 28 31 Mean ± SD 52.3 ± 23.3 53.2 ± 17.6 52.8 ± 18.5 59.2 ± 13.5 55.4 ± 17.6 Median 50 50 50 54.2 58.3 Quartiles 33.3 - 66.7 41.7 - 66.7 41.7 - 66.7 50 - 66.7 41.7 - 66.7 Range 0 - 91.7 16.7 - 83.3 0 - 83.3 33.3 - 83.3 16.7 - 91.7 Doxycycline arm Cycle 1 2 3 4 EOT* N 40 31 31 25 25 Mean ± SD 52.9 ± 18.7 48.4 ± 17 47 ± 19.5 47.3 ± 21.2 44.7 ± 20.1 Median 50 50 50 50 50 Quartiles 33.3 - 66.7 33.3 - 66.7 33.3 - 58.3 33.3 - 66.7 33.3 - 58.3 Range 16.7 - 91.7 16.7 - 83.3 0 - 83.3 0 - 83.3 0 - 83.3 *End of treatment. No relevant differences between both two treatment strategies were noticed as per the DLQI. Throughout the observation period of 8 weeks, the median values of the DLQI total score in both arms are low with a highest medium value of 4 points for both arms at the beginning of cycle 3 (except for the rise of the median at the end-of treatment assessment in the doxycycline group with a very low questionnaire compliance in this arm at that time point) (Table 5). Considering the subscales, only “symptoms and feelings” indicated major deterioration with a median of 2 points on a maximum scale of 6 (data not shown). Table 5BODY.SKIN RELATED QUALITY OF LIFE GRADED ACCORDING TO DLQI (TOTAL SCORE): Erythromycin arm Cycle 1 2 3 4 EOT* N 24 30 29 24 31 Mean ± SD 1.2 ± 2.6 5.3 ± 5.4 5.4 ± 5.6 4.4 ± 4.1 5.4 ± 5.8 Median 0 3 4 3.5 3 Quartiles 0 - 1 1.2 – 8 2 - 7 1 – 6.2 2 – 6 Range 0 - 10 0 – 25 0 - 27 0 - 14 0 – 26 Doxycycline arm Cycle 1 2 3 4 EOT* N 24 27 23 20 15 Mean ± SD 0.8 ± 1.2 4.4 ± 4.8 5.3 ± 5.2 4.9 ± 5.4 5.8 ± 4.4 Median 0 2 4 3 7 Quartiles 0 −1 1 – 5.5 2 – 7.5 1.8 – 5.2 2 – 9 Range 0 - 5 0 - 19 0- 21 1 - 22 0- 13 *End of treatment. BODY.TREATMENT ADMINISTRATION AND SAFETY: In total, 140 panitumumab administrations were recorded in the doxycycline arm (mean 3.4 per patient), and 139 in the erythromycin arm (3.6 per patient). 74% of the patients received all 4 scheduled administrations without differences in both arms. In the erythromycin arm, 33% of the patients stopped erythromycin due to insufficient activity and switched to doxycycline. Panitumumab dose reductions or dose delays were infrequent (see Supplementary Materials). As mentioned, detailed analysis of adverse events beyond skin toxicity was not within the scope of the current study. However, neither unexpected events nor frequencies were detected within the framework of the adverse events and SAE reporting in this trial. Eleven out of 41 patients (26.8%) treated within the doxycycline arm, and 7/39 patients (17.9%) in the erythromycin arm reported SAE (P =.34). BODY.ANTITUMOR EFFICACY: Regarding the overall study population, 28% of the patients experienced an objective response, while 46% had stabilization of the disease. This estimation is based on 54 patients (68%) with a valid RECIST evaluation after 8 weeks. There is no indication for an effect of the randomized assignment of the skin treatment (P =.40, Fisher's exact test; odds ratio: 1.74, 95% confidence interval: 0.49 – 6.67). BODY.DISCUSSION: This is the first randomized trial comparing an oral antibiotic prophylaxis (doxycycline) with an alternative local application (erythromycin) as preemptive skin treatment strategy against anti-EGFR targeted drugs. The primary endpoint of the present trial was the percentage of patients developing no skin toxicity grade ≥ 2 at any time during the first 8 weeks of panitumumab treatment. In the erythromycin arm, a moderate to severe skin toxicity could be prevented in only 31%, i.e. 69% suffered from skin toxicity grade ≥ 2. Thus the primary endpoint was missed and the experimental strategy (local erythromycin) would be rated as insufficient. In the reference arm, however, using doxycycline, the toxicity rate grade ≥ 2 amounted to 63%, as well, i.e. it was not lower than in the erythromycin arm. A numerical trend to a lower grade 3 toxicity with oral doxycycline can be discerned but without reaching statistical significance. Although most of the studies investigating tetracycline-family antibiotics in a preemptive setting could not demonstrate a reduction of the overall incidence of skin toxicity, they often showed a decrease of the severity compared to placebo [5, 7, 11], no treatment [8, 9], or reactive treatment [3, 9, 10, 11]. This effect could not be seen in the current trial comparing two different preemptive treatment strategies. The rates of skin toxicity grade ≥ 2 are rather high with 69% and 63%, respectively, compared to the literature. Even if considering only trials with similar settings (mCRC treated with EGFR inhibitors) the rates of grade ≥ 2 skin toxicity in the preemptive arms are considerably lower in previous studies with 20% [5], 21.3% [12] and 29% [3]) and could not be reproduced in the present trial. Until recently, the NCI CTCAE v 3.0 grading scale (published in 2006) had been used in most of the studies for evaluation of EGFR inhibitor induced skin toxicity [3, 5, 6, 8, 9, 12]. This version 3.0 included only a crude scale for severity of skin toxicity and allowed for subjective grading. The updated CTCAE v 4.0 used in the present trial provide a more thorough grading of skin toxicity taking into account that acneiform skin rash may develop only in lower percentage of skin surface. Patients that would have been classified grade 1 according to the NCI CTCAE v 3.0, could easily be regarded as grade 2 or even 3 according to the NCI CTCAE v 4.0. Thus, especially the results of the STEPP trial [3] on which the present trial was based cannot be compared with our data concerning the evaluation of skin rash. It is conceivable that the high amount of grade ≥ 2 toxicities in the present trial is due to the updated and improved grading system. Nevertheless, even the updated version 4.0 does not accurately reflect the clinical situation. One major problem of this version is that the grading is based on the affected body surface area (BSA) rather than on the (local) severity/symptoms of skin rash. An upgrading beyond the grade stipulated by the grading system is principally possible (for instance due to limited instrumental or self-care activities of daily living) but this decision is left up to the discretion of the investigator. This may of course pose problems with respect to the comparability of skin toxicity between individual investigators and between studies. In view of this, we also used an extended alternative grading system for EGFR-related skin toxicity (so called WoMo score [20]) in the present trial which takes into account also (i) the percentage of the facial area affected with rash or other dermatological adverse events as well as (ii) the type and severity of the efflorescences. Thus, the WoMo score allows for a more thorough description of the skin toxicity and enables more precise description and comparison of efficacy of different prophylactic treatments. Notably, using this grading system, we detected obvious differences between the two treatment groups. Firstly, the mean and median WoMo scores were numerically higher in the erythromycin arm. Secondly, the percentage of patients developing moderate or severe skin rash (WoMo score ≥ 20 points) was significantly higher in the erythromycin group and the time to development of moderate or severe skin toxicity was shorter in the erythromycin group (at the beginning of cycle 2 versus cycle 3). This data indicates that the experimental stepwise approach (erythromycin followed by doxycycline) was not able to at least accomplish the same results as immediate doxycycline prophylaxis. In this regard it is important to stress that the treatment intensity and adherence to panitumumab as well as the therapeutic efficacy was comparable between both arms. The analysis of the skin related quality of life using the DLQI total score shows a distinct deterioration of QoL at the beginning of cycle 2 in both arms, being rather stable thereafter in the patients remaining in the observation. The absolute amount of impact on QoL according to the DLQI seems to be rather limited with about 4 out of 30 points [14]. This is due to the finding, that only the subscale “symptoms and feelings” indicated major deterioration. All other scales were hardly affected. At the time the study was designed, the DLQI seemed to be one of the most promising questionnaires to assess skin-related QoL, not least due to the data of the STEPP trial [3]. Meanwhile, a questionnaire (FACT- EGFRI-18) is available designed to assess dermatologic symptoms associated with EGFR inhibitors [15]. It should be used for future clinical trials. In all, we were not able to find significant differences between both prophylactic treatments investigated in this trial using the NCI CTC AE v 4.0 grading system. It can be pointed out that the erythromycin arm duplicated grade 3 skin toxicity. Contrarily, by using the WoMo score we demonstrated that more patients treated with erythromycin developed moderate or severe skin toxicity at earlier time points indicating that local erythromycin should not be used as a substitute of oral tetracyclines as prophylaxis against EGFR mediated skin toxicity. We propose to use the WoMo score in addition to NCI CTCAE grading in clinical trials dedicated to study the efficacy of prophylactic or interventional measures against EGFR-mediate skin toxicity. Moreover, rash rates are high despite prophylactic treatment. These high rates beg for further research to establish improved alternative treatment options. High expectations are placed on vitamin K for use of EGFR treatment associated skin toxicity. The prophylactic use of K1 cream was demonstrated by Ocvirk [16] and – most recently – by Schimanski et al. [17]. Regarding this, the results of the EVITA trial (NCI01345526) are eagerly awaited. EVITA is the first randomized study that evaluated the addition of vitamin K to doxycycline prophylaxis. Another promising trial (NCT03051880) investigates the preventive use of topical EGF cream. Until then the prophylactic use of tetracyclines may be regarded as a standard of care in patients undergoing anti-EGFR directed treatment. BODY.MATERIALS AND METHODS: BODY.PATIENTS: Patients with metastatic colorectal cancer were eligible to participate in the study provided they met all selection criteria listed in Table 6. No study treatment or any other procedure within the framework of the trial was performed in any patient prior to receipt of written informed consent. Table 6BODY.INCLUSION AND EXCLUSION CRITERIA: Inclusion criteria: Patients with wild-type RAS (KRAS and NRAS) status of metastatic colorectal cancer treatment with panitumumab according to label RAS wild-type tested in KRAS exon 2 (codons 12/13) KRAS exon 3 (codons 59/61) KRAS exon 4 (codons 117/146) NRAS exon 2 (codons 12/13) NRAS exon 3 (codons 59/61) NRAS exon 4 (codons 117/146) Treatment with pre-emptive study medication shall begin the day before treatment start with panitumumab Willingness to cope with biweekly quality of life questionnaires Written Informed consent Aged at least 18 years ECOG Performance Status 0-2 Life expectancy of at least 12 weeks Adequate haematological, hepatic, renal and metabolic function parameters: Leukocytes > 3000/mm3 ANC ≥ 1500/mm3 Platelets ≥ 100,000/mm3 Haemoglobin > 9 g/dl Serum creatinine ≤ 1.5 x ULN Bilirubin ≤ 1.5 x ULN GOT-GPT ≤ 2.5 x ULN (in case of liver metastases GOT / GPT ≤ 5 x ULN) AP ≤ 5 x ULN Magnesium, Calcium and potassium within normal ranges (may be substituted before study entry) Exclusion criteria: Subject pregnant or breast feeding, or planning to become pregnant within 6 months after the end of treatment Subject (male or female) is not willing to use highly effective methods of contraception (per institutional standard) during treatment and for 6 months (male or female) after the end of treatment (adequate: oral contraceptives, intrauterine device or barrier method in conjunction with spermicidal jelly) Serious concurrent diseases On-treatment participation in a clinical study in the period 30 days prior to inclusion Clinically significant cardiovascular disease in (incl. myocardial infarction, unstable angina, symptomatic congestive heart failure, serious uncontrolled cardiac arrhythmia) ≤ 1 year before enrolment History of interstitial lung disease, e.g. pneumonitis or pulmonary fibrosis or evidence of interstitial lung disease on baseline chest CT scan History of HIV infection Other previous or concurrent malignancy (≤ 5 years prior to enrolment in study) except non-melanoma skin cancer or cervical carcinoma FIGO stage 0-1 if the patient is continuously disease-free Known allergic reactions on panitumumab, doxycycline or erythromycin Previous treatment with anti-cancer agents directed against EGFR (e.g. cetuximab, panitumumab, erlotinib, gefitinib, lapatinib) Skin rash existing before or due to other reasons than panitumumab treatment Other dermatologic disease that may interfere with correct grading of panitumumab induced skin rash Parallel treatment with anti-tumor agents other than panitumumab At the time the study was initiated, patients with tumors harboring no KRAS exon 2 mutations were included to be treated according to panitumumab label. Data on the negative predictive effect of mutations beyond KRAS exon 2 (i.e. KRAS exon 3-4 and NRAS exons 2-4) were first presented during the American Society of Clinical Oncology (ASCO) Annual Meeting 2013 and published later on [18, 19]. The inclusion criteria of the current trial were changed accordingly. The amendment was approved by the ethical committee on 17th January 2014. BODY.STUDY DESIGN AND TREATMENT SCHEDULE: This is a phase II, open-label, randomized, controlled, parallel-arm, multi-center study. Patients received standard tumor treatment using panitumumab in label (i.e. either as monotherapy or in combination with chemotherapy), and were randomized (1:1) to two preemptive strategies of skin toxicity prophylaxis. In order to base the trial on the best available evidence, the superior treatment arm of the phase II STEPP trial [3] was chosen as standard arm: (i) Standard arm: Skin moisturizer (in the morning on rising), sun screen (before going outdoor), doxycycline 100 mg b.i.d. starting the day before treatment start with panitumumab and for the following 8 weeks. (ii) Erythromycin arm (sequential approach): Skin moisturizer (in the morning on rising), sun screen (before going outdoor), erythromycin ointment 2% on face, hands, feet, neck, back, once daily at bedtime starting the day before treatment start with panitumumab and for the following 8 weeks; in case of skin rash ≥ grade 2 doxycycline 100 mg b.i.d. was started. The study treatment period consisted of 8 weeks (i.e. 4 administrations of bi-weekly panitumumab). BODY.STUDY OBJECTIVES: The primary objective of the study was to establish an alternative preemptive skin treatment avoiding or delaying the use of oral doxycycline in patients with wild-type (K)RAS metastatic colorectal cancer treated with panitumumab. For the primary endpoint (percentage of patients developing no skin toxicity ≥ grade 2 at any time during the first 8 weeks of treatment with panitumumab) skin toxicity was assessed using the NCI CTCAE v 4.0 criteria. Secondary objectives comprised: (i) Evaluation of skin-related (DLQI) and global quality of life (EORTC QLQ C30); (ii) assessment of skin toxicity with a different grading scale (i.e. WoMo score). BODY.STUDY ASSESSMENTS: Panitumumab was administered on a bi-weekly basis according to label (i.e. cycle length was 14 days). On day 1 of every panitumumab treatment cycle the following assessments were scheduled: (i) Physical examination including weight, ECOG status and vital signs; (ii) adverse effects according the NCI CTC Version 3.0 criteria; (iii) laboratory tests including clinical chemistry and hematology; (iv) quality of life including EORTC Quality-of-life C30, skin related quality of life with DLQI; (v) skin rash assessment including NCI CTC criteria version 4.0 and WoMo scoring. For skin toxicity grading purposes the NCI CTCAE v 4.0 was used as a primary endpoint (see Supplementary Materials). Moreover, we used the so called WoMo (Wollenberg and Moosmann) score, a thorough grading system. Briefly, the WoMo score is based on a three-part system which takes into account body involvement according to the rule of nines, percentage of facial involvement, as well as a semiquantitative description of the skin lesions by five items (erythema intensity and distribution, papulation, postulation, and scaling/crusts). The final score ranges from 0 to 100 (0 to 20 mild, 21 to 40 moderate, 41 to 100 severe acneiform eruptions) [20] (see Supplementary Materials). The assessment of skin toxicity was done by physicians trained on the NCI CTC and WoMo scores. The assessment of overall quality of life was done using the EORTC QLQ C30. Skin-related quality of life was graded according to the dermatology quality of life index (DLQI; cf: http://sites.cardiff.ac.uk/dermatology/quality-of-life/dermatology-quality-of-life-index-dlqi/). Briefly, the DLQI consists of 10 questions to assess QoL in patients with skin disorders and can be analyzed under six subscales (symptoms and feelings, daily activities, leisure, work and school, personal relationship, and treatment). It is scored on a scale of 0 to 30; higher scores indicate more QoL impairment [21]. Tumor assessments were performed according to local practice. As all antineoplastic drugs used in this trial have a well-documented adverse event profile, the assessment of adverse events beyond skin toxicity was outside the main scope of the trial. However, serious adverse events were recorded and analyzed. BODY.STATISTICAL CONSIDERATIONS: According to data derived from the STEPP trial the development of skin toxicity grade ≥ 2 can be avoided in about 70% of patients [2]. The primary hypothesis of the current trial was that a similar efficiency could be achieved by a sequential skin treatment strategy, starting with local erythromycin administration. Despite the shorter duration of the STEPP trial (6 weeks of treatment) its results had to be applicable to the 8 week situation in this trial because the primary endpoint in both trials was based on the worst grade skin toxicity which is reached after approximately 2 weeks of panitumumab treatment. The natural course of panitumumab induced skin toxicity after the climax at about 2 weeks is a steady recovery within the following weeks. The statistical calculation was based on the following premises and assumptions: According to these parameters, and using a standard single-stage phase II design by Fleming (1982), n = 37 patients evaluable for prophylactic efficacy had to be recruited. As a similar number of patients had to be recruited to the reference STEPP arm, a total number of 74 evaluable patients was required. Calculating a drop-out rate of 15% a total of 88 patients had to be included in the study. All randomized patients who received at least one application of study medication and who had at least one post-baseline assessment of the primary endpoint variable will be the full analysis set (FAS), even if they violate the selection criteria of the study. Skin toxicity (primary endpoint), clinical response and other rates were calculated, providing confidence intervals. In case of comparison between patient groups, these proportions were to be analyzed by Fisher´s exact test, X2 test or Mantel-Haenszel test (or trend test according to COCHRAN/ARMITAGE), respectively. For correlation analyses between the different quality of life and/or toxicity scores, the non-parametric test according to Spearman was preferably to be applied. The onset of grade ≥ 2 skin toxicity was to be likewise estimated by the product limit method of Kaplan-Meier and eventually compared using the logrank test. BODY.ETHICS: The study was approved by the Ethics Committee (EC), Medizinische Ethik-Kommission II of the Universitätsmedizin Mannheim, and all other participating institutions. This study was conducted in agreement with the German Drug Law (AMG), ICH Harmonized Tripartite Guideline on Good Clinical Practice, the „Verordnung über die Anwendung der Guten Klinischen Praxis bei der Durchführung von klinischen Prüfungen mit Arzneimitteln zur Anwendung am Menschen“ [22] and the Declaration of Helsinki (Tokyo, Venice, Hong Kong, Somerset West and Edinburgh amendments) [23]. BODY.SUPPLEMENTARY MATERIALS:

TITLE: Saphenous vein stripping surgical technique and frequency of saphenous nerve injury ABSTRACT.OBJECTIVES: Saphenous nerve injury is the most common complication after surgical treatment of varicose veins. The aim of this study was to establish its frequency at great saphenous vein long stripping when four methods of surgery were applied. ABSTRACT.METHODS: Eighty patients were divided into four groups depending on different stripping methods. Sensory transmission in saphenous nerve and sensory perception of shank were examined before surgery and two weeks, three and six months afterwards with clinical neurophysiology methods. ABSTRACT.RESULTS: In 36% of patients, surgeries caused the injury of saphenous nerve mainly by proximal stripping without invagination (65%, group I). Transmission disturbances ceased completely after three months in patients undergoing distal stripping with invagination (group IV), while in group I they persisted for six months in 35%. Group IV patients were the least injured and group I the most. ABSTRACT.CONCLUSION: Neurophysiological findings may suggest that distal stripping with vein invagination gives the best saphenous nerve sparing. BODY.INTRODUCTION: Varicose veins surgery is one of the most frequent operations carried out in Europe as well as one of the most frequent causes of civil-law proceedings against general and vascular surgeons, especially in West European countries, which results mainly from incidental nerve injury.1,2 The increase in medical claims forces a search for the method of varicose veins treatment that can guarantee the fewest complications possible. The fundamental approach in surgical treatment of varicose veins with great saphenous vein incompetence is the long stripping.3–5 There are several variants of the method: proximal stripping with a probe – the classical Babcock method, proximal stripping with invagination (inversion) of the vessel, distal stripping with and without invagination.6–10 The varicose veins extraction may be associated with many intra- and postsurgical complications. However, the most frequent is the saphenous nerve lesion, which is related to its anatomical path. Possible anomalies in the common anatomical passage of the saphenous nerve to the great saphenous vein should be considered during varicose veins extraction and what can foster iatrogenic injuries within higher risk zones.11 Femoral and crural portions can be distinguished in the course of saphenous nerve. The nerve runs subfascially within the femoral portion, whereas within the crural portion it pierces the crural fascia and, running epifascially together with the great saphenous vein, reaches the medial margin of the foot. The nerve supplies sensory innervation to the medial side of the lower thigh, anteromedial side of the knee joint and the leg down to medial side of the foot and hallux. During stripping of the great saphenous vein, a lesion most frequently affects the nerve trunk in region of medial malleolus and its subpatellar branch together with the medial cutaneous branches of the leg, which manifests as inaccurate feeling perception (paresthesia) in the form of tingling, numbness, electric current sensation, hypoesthesia, hyperesthesia, or a burning sensation. The aims of this study were to establish and objectivise the relation between saphenous nerve conduction disorders and varicose vein surgery as well as to compare the frequency of nerve injury in the four methods of great saphenous vein stripping. BODY.METHODS: A group of 80 patients with confirmed venous incompetence of Hach’s III and IV class by clinical (according to CEAP classification – C2 grade) and imaging (Doppler ultrasonography) examinations was enrolled into this neurophysiological prospectively gathered data.12 Patients did not report the neuropathic pain before surgery, the supplemental management with pregabalin and methylcobalamin was not applied. The patients were qualified for surgical treatment in Department of General and Vascular Surgery at Karol Marcinkowski Poznań University of Medical Sciences from 2004 to 2008 basing on the rule of consecutive cases. The neurophysiological examinations were conducted in Department of Pathophysiology of Locomotor Organs four times (before surgery-S1, two weeks after surgery-S2, three months after surgery-S3, and six months after surgery-S4). Patients aged from 20 to 50 years (42.2 years on average) were randomly divided into four subgroups (20 persons each) G1–G4 depending on the different stripping methods (G1-group after proximal stripping without invagination, G2-group after proximal stripping with invagination, G3-group after distal stripping without invagination, G4-group after distal stripping with invagination). The length of great saphenous vein stripping depended on the type of surgery. In each group, the same female to male proportion was maintained (15 females and five males). Each subgroup was operated with different stripping method. The saphenous nerve was not identified along the saphenous vein at the knee and it was not separated from it before stripping. Patients suffering from conditions that possibly affect nervous system functions, which could be polyneuropathies, multiple peripheral mononeuropathies, demyelinating diseases, neuromuscular diseases, deficiency syndromes, nervous system poisoning, advanced discopathy, extremity or vertebral column trauma, arterial diseases, episodes of superficial vein thrombosis, post-thrombotic syndrome were excluded from this study. The general methodology was based on neurophysiological examinations of saphenous nerve conduction.13–16 It was carried out four times. First time it was before the operation, in order to estimate preliminary values of neurophysiological variables (amplitude, latency, conduction velocity) in sensory conduction studies (SCV), then two weeks after the operation to establish a possible nerve injury due to surgical intervention, three months after the operation when the changes affecting conduction (swellings, hematomas, rubber bandage wearing) had disappeared and six months after the operation to assess the improvement of neurophysiological parameters in the patients with previously detected conduction disorders. The results were analyzed with reference to the control group of 50 healthy volunteers (40 females and 10 males aged 45.1 years on average) recruited from hospital workers whom the SCV reference values were acquired. The neurophysiological testing was performed with Keypoint System (Medtronic A/S, Skovlunde, Denmark). Evoked potentials were recorded at different levels of saphenous nerve conduction with standard silver chloride electrodes, using two techniques of saphenous nerve examination (distal and proximal). In the distal method, recording took place anterior to medial malleolus and stimulation 14 cm, 18 cm, 22 cm, 26 cm above, along the medial margin of tibia. In the proximal method, potentials were recorded at the midpoint between medial malleolus and the slot of the knee joint along the medial margin of tibia while stimulation was performed 14 cm, 18 cm, and 22 cm above. The ground electrode was placed halfway between the recording and stimulating electrodes. Bilateral and bipolar saphenous nerve stimulation was performed, applying supramaximal, rectangular, single electric stimuli with a frequency lower than 3 Hz, 0.2 ms duration and intensity up to 30 mA. The recordings were carried out with time base usually 2 ms, amplification 5 µV, high-pass filter at 5 Hz and low-pass filter at 3 kHz. The parameters of amplitude, latency and corresponding afferent conduction velocities were evaluated. Amplitude greater than 1 µV was considered as normal. The conduction velocity depended on the distance at which the saphenous nerve was examined. The following minimal conduction velocities were accepted: 140 mm – 38.58 m/s; 180 mm – 39.13 m/s; 220 mm – 39.85 m/s; 260 mm – 40.21 m/s for distal and 140 mm – 40.53 m/s; 180 mm – 40.86 m/s; 220 mm – 41.38 m/s for proximal method. Two methods complementary to SCV studies were used in order to study the saphenous nerve sensory function. First, it was the intensity of current versus stimulus duration curves (IC–SD) that was conducted after monopolar stimulation of skin anterior to the medial malleolus, then 14 cm and 26 cm. Second, it was the von Frey’s filaments examination (FvF) in three areas, the medial part of popliteal region, the middle 1/3 of the leg on the medial side and medial malleolus region. During each FvF examination, three measurements were performed with calibrated silicon filaments: 0.12 mm, 0.30 mm (corresponding to proper sensory perception), and 0.55 mm in diameter. Touch sensation reported in two out of three assessments was considered as positive. A positive trial with the thinnest filament indicated hyperesthesia and the completely negative trial – analgesia. The research was approved by The Bioethics Committee of Medical University in Poznań and it was performed with the ethical principles of the Helsinki Declaration. Examined subjects gave their informed consent for the examinations. All subjects were informed about the aim of study and about the course of examination. BODY.STATISTICS: In the statistical analysis of the obtained results, the Statistica programme 9.0 was used (StatSoft). The Fisher–Freeman–Halton test was used for evaluation of sample size during studies on normality distribution, similarly the McNemara when two measurements were compared (between groups) or the Cochran’s Q test when more than three measurements were considered (between stages of observations). The results were considered as statistically significant at p < 0.05. BODY.RESULTS: There was no nerve transmission abnormalities detected prior to surgery in any group. Nerve transmission abnormalities diagnosed two weeks after the operation (in 30 out of 80 patients – 37.5%) were present in 17 cases (21.25%) after three months and in 11 patients (13.75%) after six months (Table 1). Table 1.Number (percentage) of parameters changes in sensory fibers transmission of saphenous nerve (SCV studies) at four stages of observation (S1–S4) during recordings in patients from four groups with different stripping methods (G1-group after proximal stripping without invagination, G2-group after proximal stripping with invagination, G3-group after distal stripping without invagination, G4-group after distal stripping with invagination). Before surgery (S1)Two weeks after surgery (S2)Three months after surgery (S3)Six months after surgery (S4)Differences between stages of observationTotal, N = 80 0/80 (0%) 30/80 (37.5%) 17/80 (21.25%) 11/80 (13.75%) S1: S2 < 0.0001 S1: S3 < 0.0001 S1: S4 = 0.0010 S2: S3 = 0.0008 S2: S4 < 0.0001 S3: S4 = 0.0412 G1, N = 20 0/20 (0%) 13/20 (65%) 10/20 (50%) 7/20 (35%) S1: S2 = 0.0002 S1: S3 = 0.0020 S1: S4 = 0.0156 S2: S4 = 0.0412 G2, N = 20 0/20 (0%) 7/20 (35%) 4/20 (20%) 2/20 (10%) S1: S2 = 0.0156 G3, N = 20 0/20 (0%) 6/20 (30%) 3/20 (15%) 2/20 (10%) S1: S2 = 0.0312 G4, N = 20 0/20 (0%) 4/20 (20%) 0/20 (0%) 0/20 (0%) Differences between groups G1:G4 = 0.0095 G1:G3 = 0.0407 G1:G4 = 0.0083 G1:G4 = 0.0004 Statistically significant differences at p < 0.05 are marked bold. In three groups of patients (G1–G3), a statistically significant (p < 0.05) saphenous nerve lesion was identified two weeks after the surgery. In group G1 (proximal stripping without invagination), it occurred in 65%, in group G2 (proximal stripping with vein invagination) in 35%, and in group G3 (distal stripping without vein invagination) – in 30%. Best results of treatment were observed in group G4 (distal stripping with vein invagination) where no statistically significant consequences of nerve injury were detected. Six months after the operation sensory conduction disturbances were identified in a remarkable number of patients (seven out of 20 that is 35%) but only in group G1 (Table 1). Among the patients with disturbances of nerve transmission in the saphenous nerve fibers, the most frequent observation was slowing down of the conduction velocity with proper amplitude parameters (Figure 1). The results of SCV indicating deficiencies in sensory transmission are related with the results of IC–SD as well as with the von Frey filaments examination, which are presented in Tables 2 and 3. Figure 1.Examples of neurophysiological SCV recordings from saphenous nerve following electrical stimulation at distances shown on the right part of figure. Recordings were obtained in one of the patients 3 months after proximal stripping without invagination. Calibrations for amplification (µV) and time base (ms) as well as intensity of stimulation (mA) are shown in upper part of figure. Note proper values of potentials amplitudes (marked with crosses) but their prolonged latencies (marked with arrows) influenced the conduction velocities of nerve impulses in sensory fibers. Table 2.Number (percentage) of changes in sensory perception (IC–SD studies) of saphenous innervation at four stages of observation (S1–S4) during recordings in patients from four groups with different stripping methods (G1-group after proximal stripping without invagination, G2-group after proximal stripping with invagination, G3-group after distal stripping without invagination, G4-group after distal stripping with invagination). Before surgery (S1)Two weeks after surgery (S2)Three months after surgery (S3)Six months after surgery(S4)Differences between stages of observationTotal, N = 80 0/80 (0%) 49/80 (61.25%) 22/80 (27.5%) 12/80 (15%) S1: S2 < 0.0001 S1: S3 < 0.0001 S1: S4 < 0.0001 S2: S3 < 0.0001 S2: S4 = 0.0060 S3: S4 = 0.0040 G1, N = 20 0/20 (0%) 15/20 (75%) 11/20 (55%) 7/20 (35%) S1: S2 < 0.0001 S1: S3 = 0.0009 S1: S4 = 0.0156 S2: S4 = 0.0133 G2, N = 20 0/20 (0%) 14/20 (70%) 5/20 (25%) 2/20 (10%) S1: S2 < 0.0001 S2: S3 = 0.0076 S2: S4 = 0.0015 G3, N = 20 0/20 (0%) 11/20 (55%) 4/20 (20%) 2/20 (10%) S1: S2 = 0.0010 S2: S3 = 0.0233 S2: S4 = 0.0076 G4, N = 20 0/20 (0%) 9/20 (45%) 2/20 (10%) 1/20 (5%) S1:S2 = 0.0039 S2:S3 = 0.0233 S2:S4 = 0.0133 Differences between groups G1:G3 = 0.0483 G1:G4 = 0.0436 G1:G4 = 0.0054 Statistically significant differences at p < 0.05 are marked bold. Table 3.Number (percentage) of sensory perception disturbances (von Frey filaments examination) for saphenous innervation at four stages of observation (S1–S4) during recordings in patients from four groups with different stripping methods (G1-group after proximal stripping without invagination, G2-group after proximal stripping with invagination, G3-group after distal stripping without invagination, G4-group after distal stripping with invagination). Before surgery (S1)Two weeks after surgery (S2)Three months after surgery (S3)Six months after surgery (S4)Differences between stages of observationTotal, N = 80 0/80 (0%) 38/80 (47.5%) 24/80 (30%) 15/80 (18.75%) S1: S2 < 0.0001 S1: S3 < 0.0001 S1: S4 < 0.0001 S2: S3 = 0.0005 S2: S4 < 0.0001 S3: S4 = 0.0076 G1, N = 20 0/20 (0%) 15/20 (75%) 12/20 (60%) 9/20 (45%) S1: S2 < 0.0001 S1: S3 = 0.0005 S1: S4 = 0.0039 S2: S4 = 0.0412 G2, N = 20 0/20 (0%) 10/20 (50%) 4/20 (20%) 2/20 (10%) S1: S2 = 0.0020 S2: S3 = 0.0412 S2: S4 = 0.0133 G3, N = 20 0/20 (0%) 6/20 (30%) 5/20 (25%) 3/20 (15%) S1: S2 = 0.0316 G4, N = 20 0/20 (0%) 7/20 (35%) 3/20 (15%) 1/20 (5%) S1:S2 = 0.0156 S2:S4 = 0.0412 Differences between groups G1:G3 = 0.0103 G1:G2 = 0.0224 G1:G2 = 0.0309 G1:G4 = 0.0248 G1:G4 = 0.0007 G1:G4 = 0.0084 Statistically significant differences at p < 0.05 are marked bold. In postoperative observations, the significant saphenous nerve sensory perception abnormalities were detected in each group of patients based on results of IC–SD and von Frey filaments studies. Three and six months after the varicose veins extractions there was detected similar, moderate improvement of sensory perception parameters in groups G2 and G3. The best results of saphenous nerve regeneration were observed in G4 (one out of 20 patients – 5% in both IC–SD and FvF studies) and the worst in G1 (seven out of 20 patients – 35% in IC–SD examinations and nine out of 20 patients – 45% in FvF studies) groups. BODY.DISCUSSION: Saphenous nerve injury is known for a long time to be a potential complication of the saphenous vein long stripping.6–10,13–24 The proximity of the vein and nerve, especially at the level of shank, results in injuries during the vein resection,25–29 especially in the patients with advanced varicose veins in this area caused by a large number of insufficient perforators. Additionally, in the patients suffering from long-lasting varicose veins, it can lead to an accretion of the widened-vein, providing the saphenous nerve neuropraxy. This pathology also facilitates in injury of the nerve fibers during the operation. The sensory disorders associated with an injury to the nerve trunk or its branches, occurring after the varicose veins surgical treatment, can be either temporary or constant.23,26 The results presented in this paper, referring to the saphenous nerve injury during the varicose veins surgical treatment using the saphenous vein long stripping, correlate with the results that are to be found in publications devoted to the subject.18,19,21 Many authors tried to determine the frequency of this complication. It varies from 5.9% to 72% and depends to a large extent on the applied stripping technique.17–19,21,23–26 In the study presented, the most numerous saphenous nerve injuries were identified after the proximal long stripping without vein invagination (the Babcock method). Such complications were reported in 20–50% of patients treated with classical stripping of the Babock type.27 It is joined to the anatomical relation of the saphenous vein and the saphenous nerve together with its divisions. Anatomically, the nerve branches form a reverted V shape, which facilitates their injury by the probe’s head, especially while proximal pulling of the vein.17,18,28 Invagination of the vein as well as the change in the direction of its removal from proximal to distal caused the reduction in the number of neurological complications related to the saphenous nerve injury.8,19 The proximal stripping with invagination of the vessel and the distal stripping without invagination occurred to be comparable in terms of the number of postsurgical sensory disorders in the saphenous nerve innervation area. The best effects were achieved with distal stripping with vein invagination.19 From the results of this study, it can be concluded that this method is the least invasive for the saphenous nerve. All the conduction abnormalities that were objectively identified in SCV studies two weeks after the surgery were temporary and normalized at three months after the operation. Part of the patients subjectively still suffered from some sensory disorders, which was confirmed in FvF and IC–SD examinations perhaps due to the injury in small branches of saphenous nerve. Based on the results of neurophysiological findings provided in this study, we suppose that compilation of distal stripping with vein invagination gives the best saphenous nerve sparing during varicose veins extraction. Transient changes in nerve transmission observed two weeks after surgery can be due to the temporary neuropraxia caused by edema, hematoma, inflammatory processes, or simply by the mechanical irritation during operation. A large number of neurological complications after the varicose veins surgery in lower extremities with the use of the saphenous vein stripping make alternative means of treatment of the saphenous vein incompetency worth consideration.30–34 One of these alternative methods is the short stripping to the level of knee joint with additional phlebectomy of the varicose veins below this level.24 It should be remembered, however, that in spite of more and more innovative methods of the varicose veins treatment, the long stripping is still the most common procedure and a first-choice therapy for the saphenous vein incompetence.3–5 Even if the sensory disorders developed after the surgical removal of the whole vein can persist in some patients, the advantages of varicose veins resection much exceed the inconveniences resulting from the potential neurological complications. Nerve injuries also might appear after thermal ablation associated with varicose veins surgeries and there is a need for a similar like presented study of patients having various endovenous treatments. The limited size of this study seems to be a weakness of the presented report, but its results may suggest that neurophysiological testing is valuable for evaluation of the treatment efficiency after varicose veins surgeries. BODY.CONCLUSIONS: Varicose veins surgery in the lower limbs using the technique of the saphenous vein long stripping significantly impacts the sensory conduction in the saphenous nerve. Distal stripping with vein invagination seems to be the least invasive for the saphenous nerve fibers, which may be a consequence of the anatomical relation of the saphenous nerve and vein. Proximal stripping without vein invagination leads to the most numerous neurological complications detected two weeks after the operation as well as three and six months after the procedure, when the changes possibly affecting the nerve transmission had ceased.

TITLE: A randomized two way cross over study for comparison of absorption of vitamin D3 buccal spray and soft gelatin capsule formulation in healthy subjects and in patients with intestinal malabsorption ABSTRACT.BACKGROUND: Vitamin D deficiency has been proposed to contribute to the development of malabsorption diseases. Despite this, the vitamin D status of these patients is often neglected. The objective of the present work was to compare the absorption of vitamin D3 through the oral route by comparing a 1000 IU soft gelatin capsule and a 500 IU buccal spray (delivering 1000 IU in two spray shots) in healthy subjects and in patients with malabsorption disease. ABSTRACT.METHODS: An open label, randomized, two-periods, two-way cross over study was conducted, first in healthy subjects (n = 20) and then in patients with malabsorption syndrome (n = 20). The study participants were equally divided and received either of the treatments (buccal spray, n = 7; soft gelatin capsule, n = 7; control, n = 6) in Period I for 30 days. After washout of another 30 days, the treatments were changed in crossover fashion in Period II. Fasting blood samples were collected to measure baseline 25-hydroxyvitamin D [25(OH)D] levels in all participants at day 0 (Screening visit), day 30 (completion of period I), day 60 (end of wash out and initiation of period II) and day 90 (completion of period II). Safety was evaluated by hematology and biochemistry analyses. Statistical analyses was performed using differences of mean and percentage change from baseline of 25(OH)D levels between two formulation by two tailed Paired t-test with 95 % confidence interval. ABSTRACT.RESULTS: In healthy subjects, the mean increase in serum 25(OH)D concentration was 4.06 (95 % CI 3.41, 4.71) ng/ml in soft gelatin capsule group and 8.0 (95 % CI 6.86, 9.13) ng/ml in buccal spray group after 30 days treatment (p < 0.0001). In patients with malabsorption disease, the mean increase in serum 25(OH)D concentration was 3.96 (95 % CI 2.37, 5.56) ng/ml in soft gelatin capsule group and 10.46 (95 % CI 6.89, 14.03) ng/ml in buccal spray group (p < 0.0001). ABSTRACT.CONCLUSION: It can be concluded from the results that the buccal spray produced a significantly higher mean serum 25(OH)D concentration as compared to the soft gelatin capsule, in both healthy subjects as well as in patients with malabsorption syndrome over a period of 30 days administration in a two way cross over study. Treatments were well tolerated by both subject groups ABSTRACT.TRIAL REGISTRATION: CTRI/2013/06/003770 BODY.INTRODUCTION: Vitamin D is essential for active intestinal calcium absorption and plays a central role in maintaining calcium homeostasis and skeletal integrity. It is derived mainly from cutaneous synthesis in the presence of ultraviolet sunlight while dietary intake constitutes a minor fraction [1]. Vitamin D deficiency is a common problem through the world [2, 3] and is assessed by low serum concentration of the major circulating metabolite 25-hydroxyvitamin D (25(OH)D) [4, 5]. The prevention of vitamin D deficiency and insufficiency remains a priority of international health services [6–8]. Vitamin D deficiency has been proposed to contribute to the development of intestinal bowel diseases like Crohn's disease, steatorrhea and ulcerative colitis [9]. Conversely, people who have such illnesses have a reduced absorption of vitamin D3 through the intestine [10, 11]. In addition, osteomalacia occurs in patients with a wide variety of disorders affecting the stomach and small bowel, especially when associated with steatorrhea. The pathogenesis of this osteomalacia has in part been explained by malabsorption in vitamin D [9]. Earlier reports show that orally administered tritiated vitamin D3 was malabsorbed in patients with celiac disease, biliary obstruction or pancreatic disease [12]. The pathogenesis of vitamin D deficiency in these patients remains unclear but it is thought to result primarily from fat soluble vitamin malabsorption due to the presence of intestinal disease conditions Despite vitamin D malabsorption in patients with gastrointestinal or liver disease, the vitamin status of these patients is often neglected. Although vitamin D supplements are often prescribed, adequate absorption of these formulations has not been documented [10]. Vitamin D is known to be liposoluble, and its relative bioavailability could result in unfavorable conditions when administered in solid form (capsule), since the process of its release is a factor limiting the rate of absorption, bearing in mind that bioavailability is related not only to the pharmaceutically active molecules, but also, to the formulation and excipients used. Vitamin D3 taken by oral route (peroral delivery) is absorbed in the intestine, where the lining of the digestive tract is aqueous in nature. Therefore vitamin D3, a fat-soluble molecule, in order to be absorbed, must be made water soluble in the intestine. This is accomplished in two steps: emulsification of vitamin D3 in the intestinal lumen, through the action of bile salts, forming small droplets which are dispersed and incorporated into micelles-complex aggregates formed by the interaction of free fatty acids, monoglycerides, and bile salts. Micelles are sufficiently water-soluble to access the intestinal brush border where upon the vitamin D3 content is released and then absorbed [13]. When sprayed inside the mouth, the fine micro sized droplets of vitamin D3 are believed to be quickly and completely absorbed through the buccal mucosa into the numerous capillaries and veins lying close to the tissue surface [13]. Considering the possibility of reduced vitamin D absorption in healthy subjects and even in patients with malabsorption syndrome, a buccal spray formulation was developed. Therefore, the objective of the present work was to compare the absorption of vitamin D3 through the oral route (soft gelatin capsule form) and buccal spray in healthy subjects and patients with intestinal malabsorption syndrome. BODY.METHODS: BODY.STUDY DESIGN AND PATIENTS: An open label, randomized, two-periods, two-way cross over study was conducted, first in healthy subjects and then in patients with malabsorption syndrome, with a similar study design except the presence of disease status in patients with malabsorption syndrome. After approval from the Spandan-IEC ethics committee (registration no: ECR/67/Indt/GJ/2013), the informed consent of study participants were taken and the formulation was administered for 30 days in period I where half of the subjects and patients (collectively participants) received capsule formulation and half of the participants received buccal spray formulation. After completion of treatment in period I, all participants were given 30 days wash out before initiating period II where treatment has changed in cross over fashion, i.e. the participants who received capsule formulation in period I have received buccal spray in period II and vice versa. The clinical study was registered in a centralized clinical trial registry of India (CTRI) before initiating the enrollment of the first patient in the study (CTRI/2013/06/003770). The inclusion criteria were as follows: subjects of either sex between 18 and 65 years of age, with a Body Mass Index (BMI) between 18.0 and 30.0 kg/m2, and the ability to comply with study procedures in the opinion of the investigators. For healthy subjects: no history of liver, kidney or cardiovascular disease, or of any other medical conditions or medications likely to affect vitamin D3 absorption or metabolism. For patients with malabsorption syndrome: confirmed diagnosis of any one of the following malabsorption disease conditions like ulcerative colitis, Crohn’s disease or steatorrhea. Patient with history of above diseases, who are on therapy, were selected for the screening. In these patients, malabsorption syndrome was diagnosed by clinical symptoms like abdominal pain, vomiting, diarrhea, and subcutaneous fat loss together with blood tests like haematology and biochemistry. Stool examination was also performed for all patients to confirm rectal bleeding, presence of occult blood, infectious organisms, or fat. Finally, colonoscopy was performed in all patients to objectively assess the extent of inflammation to confirm the diagnosis. The exclusion criteria were as follows: systemic inflammatory or malignant disease; hepatic or renal failure; uncontrolled hypo- or hyperthyroidism, or the use of drugs that are known to affect bone metabolism such as bisphosphonates, glucocorticoids and anti-convulsants. A pregnant or desired to be pregnant woman during study period was also excluded. BODY.DATA COLLECTED AT BASELINE: Each participant completed a self-administered questionnaire before enrollment. During completion of the questionnaire, they had the possibility to ask for assistance (i.e. clarification of question or any other issue) from one of the project leaders. The questionnaire included questions about usual intake of vitamin D containing foods, clothing and sun exposure habits, as well as height & weight, date of birth, and education. Body mass index was derived as wt/ht2 (kg/m2). Blood pressure was measured through sphygmomanometer and vitals (heart rate, body temperature) were also recorded for safety purposes. BODY.COLLECTION AND ANALYSES OF BLOOD SAMPLES: Fasting blood samples were collected to measure baseline 25(OH)D levels in all the participants at day 0 (Screening visit), day 30 (completion of period I), day 60 (end of wash out and initiation of period II) and day 90 (completion of period II). Blood samples were centrifuged (15 min; 2000 g at 4 °C) within 30 min of blood collection and separated serum samples were immediately frozen. Serum samples were stored at −20 °C until analyzed. Serum 25(OH)D levels were measured by Electrochemiluminescence (ECLIA) assay method. This assay was carried out through quantitative determinations of total 25-hydroxyvitamin D in serum samples using a standard kit available from Roche diagnostics GmbH, Germany. All analyses were done in a central independent clinical analysis laboratory (APL Institute of Clinical Laboratory & Research Pvt. Ltd., Ahmedabad, India). The kit has a limit of detection of 3 ng/mL and has a linearity of 0.0 to 60.0 ng/mL. The intra-assay and inter-assay co-efficient of variation were 4 % and 6 %, respectively. Elecsys e-immunoassay analyzers were used for this assay. Safety parameters were evaluated including hematology analyses (complete blood counts), biochemical analyses (serum creatinine, total bilirubin, urea, SGOT, SGPT, alkaline phosphates, calcium) and urine was collected for urine routine and microbiological analyses at screening visit (day 0) and at the end of period II visit (day 90). BODY.INTERVENTION RANDOMIZATION AND COMPLIANCE: BODY.RANDOMIZATION AND GROUP ALLOCATION:: The participants were enrolled at two different hospital sites in India; one physician’s site where all healthy subjects were recruited and a gastroenterologist’s site where all patients with intestinal malabsorption were recruited. Out of the forty-eight participants who met the eligibility criteria, forty (twenty healthy and twenty patients) had agreed to participate and were found eligible, signed a written consent form, and completed a self administered questionnaire concerning usual diet and sun exposure. Subsequently, a venous blood sample was drawn, and a participant received a sealed, non-transparent envelope with the allocated treatment intervention. The randomization procedure was performed beforehand by a statistician by block randomization with blocks of two to one ratio, where the first two participants were randomly given each treatment and a third participant served as a control and didn’t receive any treatment. The participants were allocated to the interventions as they visited the clinic i.e. the first subject was allocated to buccal spray group, second to soft gelatin capsule group and third to the control group. This was done in order to distribute the participants equally in the two interventions (Group I and Group III) and half of the participants served as a control (Group II and Group IV). Twenty healthy subjects enrolled for the study were recruited at a physician’s site. They were randomized and enrolled into group I and II: fourteen subjects were enrolled for vitamin D3 treatment and labeled as group I (healthy subjects), while every third subject (total six subjects) were not given any treatment (labeled as group II) and acted as the control for group I. The twenty patients with confirmed diagnosis of malabsorption syndromes (nine with ulcerative colitis, four with Crohn’s disease and seven with steatorrhea) were recruited at a gastroenterologist’s hospital site. Fourteen subjects were enrolled for vitamin D3 treatment and labeled as group III (patients with malabsorption syndrome), while every third subject (total six subjects) were not given any treatment (labeled as group IV) and acted as the control for group III. All patients continued to take their treatment for ulcerative colitis, Crohn’s disease and steatorrhea as prescribed by gastroenterologists. Treatment allocation was assigned to each participant according to their number of sequence of attendance at the blood sampling. Treatment allocation was concealed in envelopes numbered in ascending order that they met. Study personnel involved in recruitment and data collection were blinded to the participant’s treatment allocation. All participants were instructed to maintain their routine lifestyle including diet habits and sun exposure during the entire study period, to minimize interference with the daily routine. BODY.INTERVENTION: The buccal spray and soft gelatin capsules containing vitamin D3 were supplied by Pharma Base, India (a subsidiary of Pharma Base SA, Switzerland). The soft gelatin capsule formulation was purchased from the Indian market. The analysis of both the formulations was done in triplicate according to the method described in European Pharmacopeia at an independent analytical laboratory (Oasis Testing House, Ahmedabad, India). The soft gelatin capsule formulation had a label content of 1000 IU per capsule and the buccal spray formulation had a label content of 500 IU/spray shot. All participants in group I & III were randomized to receive either the vitamin D3 buccal spray (2 sprays each of 500 IU) or soft gelatin capsule containing vitamin D3 (1000 IU) for 30 days. Of the fourteen subjects in each group, half received buccal spray and half received soft gelatin capsule. This was considered as period I of the study. After the completion of the 30-day treatment, all participants were given a 30-day washout. The next treatment in period II was changed in a crossover fashion; those participants who had received the buccal spray formulation (vitamin D3) in period I received the soft gelatin capsule formulation in period II and vice versa. The treatment in period II continued in group I and group III participants for the next 30 days. The detailed study flow chart is described in Fig. 1.Fig. 1Study flow chart BODY.COMPLIANCE: All participants were instructed to take the two buccal sprays and one soft gelatin capsule every day irrespective of the period of the study. Formulation for 7 days was handed over to each participants under group I & III at baseline, along with the compliance form. The participants were instructed to mark the intake and time of intake of number of spray shots or capsule of each day of the study period, as well as to note any extraordinary event that occurred during the period (forgetting to take a spray shot or capsule). If forgetting to take spray shot or capsule, the participants were instructed to take a double dose on the following day, in order to take altogether 60 spray shots or 30 capsules during the study periods. The participants were recommended to take spray shots or capsules after the main meal of the day. However, participants were not instructed to standardize meals or time between meals. All participants were also instructed not to change their daily routine, meal habits, as well as their sun exposure. This was done to minimize interference with daily routine and thus maximizing compliance with taking study medication. All participants in group I and III were instructed to visit the site every week to check compliance with the intake of study medication. Participants in control groups (Group II and Group IV) were instructed to visit their respective clinic after 30 days for their blood sample collection. All participants who received treatment were also instructed to note any adverse events during the entire 90 days of the study which included period I, washout period and period II in a separately given adverse event recording form. BODY.STASTICAL ANALYSIS: BODY.SAMPLE SIZE: The sample size of this crossover study was based on the changes in 25(OH)D levels where goal was to detect: 1) percent increase between pre-dose and post-dose levels within treatments, and 2) to have significant difference of percent change in 25(OH)D levels between the treatments. To achieve type-I error rate of less than 5 % (2 tailed), a sample size of 12 subjects in buccal spray group (healthy or patients) was sufficient to provide a statistical power of 80 % to detect a clinically significant mean difference of 5 % in 25(OH)D levels. BODY.DEMOGRAPHIC AND BASELINE CHARACTERISTIC: A descriptive statistics was applied for demographics; age (years), weight (kg), body mass index (kg/m2); and were presented as mean ± standard deviation. BODY.EFFICACY ANALYSIS: Statistical analyses were performed using GraphPad Prism 5, version 5.03 (GraphPad Software, Inc., CA, USA). Differences of mean and percentage change from baseline of 25(OH)D levels between two formulation groups, soft gelatin capsules and buccal spray, were evaluated using two tailed Paired t-test with 95 % confidence interval separately for both healthy subjects and patients with intestinal malabsorption. For comparison between control group and either of the treatments, unpaired t-test with 95 % confidence interval was used. BODY.SAFETY ANALYSIS: The number and proportion of subjects with changes in laboratory value (change from baseline to end of study visit for complete blood count, serum creatinine, total bilirubin, urea, SGOT, SGPT, alkaline phosphates and calcium) was summarized and the difference was analyzed by chi –square test from normal to abnormal. BODY.RESULTS: BODY.BASELINE CHARACTERISTICS:: In total, thirty eight individuals, thirteen healthy individuals with six control and thirteen patients with malabsorption syndrome with six control, fulfilled the eligibility criteria and completed the study (Fig. 1). Two participants (one healthy subject and one patient) reported vitamin D3 intake compliance of less than 90 % and therefore were not considered for final analyses. In healthy subjects, at baseline, 85 % (n = 12) of the subjects had serum 25(OH)D concentration between 10 and 30 ng/ml, and one individual each (7.14 %) had serum 25(OH)D concentration >30 ng/ml and <10 ng/ml each in the oral soft gelatin capsule group. While in the buccal spray group, at baseline all subjects had serum 25(OH)D concentration between 10 and 30 ng/ml. Similarly, in patients with malabsorption disease who received oral soft gelatin capsules, at baseline 8 (57.14 %) patients had serum 25(OH)D concentration below 10 ng/ml, 5 (35.71 %) patients had serum 25(OH)D concentration between 10 and 20 ng/ml, and one individual (7.14 %) had serum 25(OH)D concentration above 20 ng/ml. However, in the buccal spray group, at baseline 9 (64.28 %) patients had serum 25(OH)D concentration below 10 ng/ml and 5 (35.71 %) patients had serum 25(OH)D concentration between 10 and 20 ng/ml. There were no striking differences in baseline characteristics between healthy individuals or patients with malabsorption disease with their corresponding control groups (Table 1).Table 1Demographic Data for Healthy Subjects and Patients with malabsorption syndrome ParametersHealthy Subjects Group IHealthy Subjects Control Group IIPatients with malabsorption syndrome Group IIIPatients with malabsorption syndromeControlGroup IVN 14 6 14 6 Sex Male = 7, Female = 7 Male = 3, Female = 3 Male = 7, Female = 7 Male = 3, Female = 3 Age (Yrs) 36.21 ± 9.97 34.00 ± 6.42 39.93 ± 11.65 44.17 ± 5.56 (Range) (25–60) (25–42) (26–63) (38–53) Height (cms) 159.86 ± 13.43 161.33 ± 14.12 162.29 ± 8.54 164.33 ± 8.55 BMI 23.39 ± 3.88 21.40 ± 2.39 21.48 ± 2.82 23.64 ± 3.02 All values are expressed in Mean ± SD; N-number subjects in each group BODY.EFFECT OF INTERVENTION: The control groups in each subject population were compared with their corresponding treatment group. The control groups for healthy subjects as well as patients with malabsorption syndrome showed no change in 25(OH)D concentration over a period of 30 days. The mean baseline levels of 25(OH)D in healthy subjects was 18.25 ng/ml and in patients with malabsorption syndrome was 11.7 ng/ml. These mean levels remained at 18.06 ng/ml and 12.52 ng/ml after 30 days in healthy subjects and patients with malabsorption syndrome respectively (Fig. 2), which was statistically non-significant. When control group in healthy subjects was compared with their corresponding treatment groups, it was found that the difference of mean between control group and buccal spray group was 7.47 (95 % CI, 5.27, 9.67) which was significant (p < 0.05), and the same between control group and soft gelatin capsule group was 3.53 (95 % CI, 1.79, 5.28) which was also statistically significant (p < 0.05). Similarly in patients, the difference of mean between control group and buccal spray group was 8.53 (95 % CI, 2.74, 14.31) which was significant (p < 0.05), while the difference of mean between control group and soft gelatin capsule group was 2.03 (95 % CI, −1.44, 5.50) which was statistically not significant. This shows that buccal spray was more effective to increase mean 25(OH)D levels as compared to oral soft gelatin capsule. Table 2 describes these data.Fig. 2Mean 25(OH)D level in study subjects Table 2Comparison of 25(OH)D in control group and their corresponding treatment groups after 30 days ComparisonDifference of mean95 % CIP ValueHealthy – Buccal Spray vs. Control 7.47 5.27 to 9.67 <0.0001 Healthy – Soft Gelatin Capsule vs. Control 3.53 1.79 to 5.28 0.0005 Patients – Buccal Spray vs. Control 8.53 2.74 to 14.31 0.0064 Patients – Soft Gelatin Capsule vs. Control 2.03 −1.44 to 5.50 0.2338 The efficacy of buccal spray and soft gelatin capsule to increase the 25(OH)D levels after 30 days of administration was evaluated and compared with each other. After 30 days of administration, overall mean serum 25(OH)D concentration in healthy subjects was 22.75 (sd 6.75) ng/ml as compared to baseline value of 18.69 (sd 5.88) ng/ml in the soft gelatin capsule group, with the mean increase of 4.06 (95 % CI 3.41, 4.71) ng/ml (Table 3). On the other hand, in the buccal spray group, the mean serum 25(OH)D concentration was 26.91 (sd 5.72) ng/ml as compared to baseline value of 18.91 (sd 4.3) ng/ml, with the mean increase of 8.0 (95 % CI 6.86, 9.13) ng/ml (Fig. 2). The difference in mean increase between both the groups was 3.95 (95 % CI 3.19, 4.69) which was statistically significant (p < 0.0001).Table 3 ParametersMean baseline value in ng/mlMean value in ng/ml after 30 day of treatmentDifference of meanPercentage mean increase from baselineHealthy Subjects Soft gelatin Capsule Formulation  Mean 18.69 ± 5.88 22.75 ± 6.75 4.06 21.72 %  Range 9.25, 30.05 12.75, 35.52 2.5, 5.6 15.36, 37.79  n 13 13 13 13  95 % CI 15.14 to 22.24 18.67 to 24.42 3.41 to 4.71 16.42 to 24.42 Buccal Spray Formulation  Mean 18.91 ± 4.3 26.91 ± 5.72 7.995 42.99 %  Range 13.36, 26.82 19.6, 38.5 4.5, 11.67 29.21, 68.71  n 13 13 13 13  95 % CI 16.31 to 21.51 23.45 to 30.36 6.86 to 9.13 37.19 to 48.79 Patients with intestinal malabsorption syndrome Soft gelatin Capsule Formulation  Mean 11.01 ± 6.43 14.97 ± 9.01 3.965 36.02 %  Range 2.9, 26.5 4.6, 36.89 1.06,10.39 24.62,58.73  n 13 13 13 13  95 % CI 7.12 to 14.89 9.52 to 20.42 2.37 to 5.56 30.42 to 41.62 Buccal Spray Formulation  Mean 10.01 ± 4.29 20.47 ± 7.89 10.46 117.8 %  Range 4.6, 18.85 9.8, 34.64 4.25, 27.44 61.31,381.1  n 13 13 13 13  95 % CI 7.42 to 12.6 15.7 to 25.24 6.89 to 14.03 64.71 to 170.8 When calculated from baseline, the mean percentage change in serum 25(OH)D concentration in healthy subjects after 30 days treatment with soft gelatin capsule was 21.72 % (95 % CI 16.42, 24.42), while the same in buccal spray group was 42.99 % (95 % CI 37.19, 48.79) with a mean difference of 20.42 % (95 % CI 16.42, 24.42) between two groups (p < 0.0001) (Table 3). A total of 11 (85 %) subjects now had serum 25(OH)D concentration between 10 and 30 ng/ml, and two individuals (15.38 %) now had serum 25(OH)D concentration >30 ng/ml in the soft gelatin capsule group. However, in the buccal spray group, 10 (75.88 %) subjects had serum 25(OH)D concentration between 10 and 30 ng/ml and 3 (23.1 %) subjects now had serum 25(OH)D concentration >30 ng/ml. Similarly in patients with malabsorption syndrome, overall mean serum 25(OH)D concentration after 30 days of administration of soft gelatin capsule was 14.97 (sd 9.01) ng/ml as compared to baseline value of 11.01 (sd 6.43) ng/ml, with the mean increase of 3.96 (95 % CI 2.37, 5.56) ng/ml. While in buccal spray group, mean serum 25(OH)D concentration was 20.47 (sd 7.89) ng/ml as compared to baseline value of 10.01 (sd 4.29) ng/ml, with the mean increase of 10.46(95 % CI 6.89, 14.03) ng/ml (Fig. 2). The difference in mean increase between both the groups was 6.50 (95 % CI 3.78, 9.22) which was statistically significant (p < 0.0001). The mean percentage change in serum 25(OH)D concentration in patients with malabsorption syndrome after 30 days treatment with soft gelatin capsule was 36.02 % (95 % CI 30.42, 41.62), while the same value in buccal spray group was 117.8 % (95 % CI 64.71, 170.8) with a mean difference of 81.75 % (95 % CI 29.80, 133.7) between the two treatments (p < 0.005). A total of four (31 %) subjects now had serum 25(OH)D concentration below 10 ng/ml, eight (61 %) subjects now had serum 25(OH)D concentration between 10 and 30 ng/ml, and one individual (7.7 %) had serum 25(OH)D concentration above 30 ng/ml. However, in the buccal spray group, only 7.7 % (1 subject) now had serum 25(OH)D concentration below 10 ng/ml, ten (76.9 %) subjects now had serum 25(OH)D concentration between 10 and 30 ng/ml and two (15.4 %) subjects now had serum 25(OH)D concentration more than 30 ng/ml (Table 3). BODY.STATISTICAL CONSIDERATIONS WITH RESPECT TO PERIOD, SEQUENCE AND POWER:: Statistical analyses were performed using SAS v9.2 (SAS Institute Inc, Cary, NC, USA) for the post-hoc evaluation of sequence and period effect. The statistical method adopted for this analysis was period + sequence + Subject (sequence) + treatment. It was observed that there is no significant difference in sequence effect in healthy subjects (p = 0.5251) and in patients with malabsorption syndrome (p = 0.0532). It was also revealed that there is no statistically significant period effect in healthy subjects (p = 0.6920) as well as in patients with malabsorption syndrome (p = 0.0715). The post-hoc power analyses with intra-subject variability were also derived for both the group of subjects. In healthy subjects, statistical power obtained was 99.42 with an 11.81 % intra-subject variability. While, in patients with malabsorption syndrome it was 81.62 with an intra-subject variability of 21.86 %. BODY.SAFETY EVALUATION: There were no significant changes in any of the hematology and biochemistry parameters studied. There are also no notable changes in the vitals for any of the participants. No adverse event reported after administration of the buccal spray or soft gelatin capsule formulations of vitamin D3 during entire study period in healthy or patients with malabsorption disease and hence the product is considered safe. BODY.DISCUSSION: Supplemental fat-soluble vitamin D is usually made without determination of whether oral doses are adequately absorbed. The evidence of vitamin D malabsorption (Osteomalacia, rickets, hypocalcaemia, or reduced circulating concentration of 25(OH)D) persists despite routine vitamin D supplementation in cystic fibrosis [14], Crohn’s disease [15], Intestinal resection [16–19], ulcerative colitis, liver disease [20–22] and other malabsorption syndrome [23]. Many factors are involved in the absorption of vitamin D, including gastric, pancreatic, and biliary secretions, micelle formation, and diffusion through the unstirred water layer, brush border membrane uptake, and transport out of the intestinal cell [24, 25]. As vitamin D is a relatively non-polar sterol, it must be solubilized by incorporation into a bile salt micelle solution in order to be absorbed in the aqueous phase [26]. This process is severely inhibited if there is any interruption of normal pancreatic or biliary secretion. As fat-soluble vitamins are fairly sensitive to disturbances in lipid absorption, vitamin malabsorption may occur in conditions like steatorrhea, ulcerative colitis, and Crohn’s disease. Serum concentrations of 25-hydroxyvitamin D are good indicators of long term vitamin D levels in the body but are insensitive to single doses of vitamin D and do not rise out of the normal range unless doses of vitamin D are chronically administered [27]. Considering the malabsorption in intestinal disease and a possible poor and complex absorption with oral formulations, a novel nanoemulsion formulation of buccal spray was developed where vitamin D3 is suspended in an aqueous base which can be easily absorbed through the mucosal layer of the mouth. We compared the serum concentration of vitamin D after 30 days administration with the soft gelatin capsule and the aqueous based buccal spray formulation. To the best of our knowledge, this is the first randomized two way cross over trial comparing the increase in 25(OH)D levels in healthy adults and patients with intestinal malabsorption receiving similar oral doses of two different formulations (per oral and buccal spray). The analyses of baseline levels of study participants showed that vitamin D deficiency was prevalent in both healthy subjects and patients with intestinal malabsorption syndrome. However, patients were more vitamin D deficient as compared to healthy subjects. Four weeks administration of 1000 IU per day increased mean serum 25(OH)D in all treatment groups. In healthy subjects, soft gelatin capsule increased serum 25(OH)D level by 22.5 % (Range 15.4 to 37.8 %), while the buccal spray increased serum 25(OH)D level by 43 % (range 29.2 to 68.7. Similarly, in patients with intestinal malabsorption, soft gelatin capsule increased serum 25(OH)D level by 36 % (range 24.6 to 58.7 %) and the buccal spray increased serum 25(OH)D by 117.8 % (range 61.3 to 381.1 %). The result implies that the buccal spray formulation had a significantly higher mean increase in both the subject groups, healthy subjects and patients with intestinal malabsorption syndrome. Interestingly, the mean increase was much higher in the patients group as compared to the healthy subjects group. This may be because increase in serum 25(OH)D after supplementation is known to be inversely related to baseline 25(OH)D concentration [28]. In the present study, the mean baseline levels were almost half in patients with intestinal malabsorption as compared to the baseline levels found in healthy subjects. This indicates the presence of vitamin D3 deficiency in the patient group as compared to healthy subjects. The primary and the most important source of vitamin D is sunlight. Although excessive exposure to sunlight and vitamin D have been positively associated with non-melanoma skin cancer [29], ecological studies suggest that sunlight may protect against female breast, ovarian, prostate, and colon cancer [30]. Solar UV-B exposure and the amount of exposure to sun are related inversely with cancer mortality and survival in detailed epidemiological studies [31]. Some analytical studies suggest a protective association between circulating vitamin D in blood, which is largely derived from sunlight or dietary vitamin D, and colorectal cancer and prostate cancer [30]. However, looking at the overall baseline levels of all participants in the present study, it is also advisable to increase the moderate daily sun exposure and to improve clothing apart from vitamin D3 supplementation. BODY.CONCLUSION: We conclude that the buccal spray formulation was able to increase mean serum vitamin D3 concentration significantly higher as compared to the soft gelatin capsule, in both healthy subjects (1.9 times) as well as in patients with intestinal malabsorption syndrome (2.6 times). BODY.AVAILABILITY OF SUPPORTING DATA: The data set(s) supporting the results of this article is (are) included within the article.

TITLE: Effects of a home-based intervention on diet and physical activity behaviours for rural adults with or at risk of metabolic syndrome: a randomised controlled trial ABSTRACT.BACKGROUND: This study aimed to determine whether a home-based 6-month lifestyle intervention program complemented by motivational interviewing could improve diet and physical activity behaviours in 50–69 year olds with or at risk of metabolic syndrome, residing in a disadvantaged rural Western Australian community. ABSTRACT.METHODS: Participants from the City of Albany and surrounding towns (n = 401) were recruited into a 6 month randomised controlled trial. They were screened for metabolic syndrome and randomly allocated to intervention (n = 201) or control group (n = 200). Baseline and post-test data collection for both groups included a self-report questionnaire which incorporated the Fat and Fibre Barometer and the International Physical Activity Questionnaire Short Form. The intervention group received the program materials at baseline and the control group was waitlisted. Generalised estimating equation models assessed repeated outcome measures over time. ABSTRACT.RESULTS: A total of 151 (75.1 %) intervention and 159 (79.5 %) control group participants completed post-test and were included in the analysis. After controlling for confounders, the intervention group achieved a marginally significant increase in their metabolic equivalent (MET) minutes of moderate intensity physical activity per week (p = 0.049), and significantly improved fibre intake (p < 0.001), fat intake (p = 0.003), and vegetable serves per day (p = 0.002) from baseline to post-test relative to the control group. ABSTRACT.CONCLUSION: A home-based, low-cost intervention with motivational support can effectively improve the physical activity and dietary behaviours of adults aged 50–69 years with or at risk of metabolic syndrome residing in a disadvantaged rural area. ABSTRACT.TRIAL REGISTRATION: Anzctr.org.au Identifier: ACTRN12614000512628 BODY.BACKGROUND: Metabolic syndrome is characterised by several metabolic abnormalities including raised triglyceride levels, reduced high density lipoprotein (HDL) cholesterol, hypertension, hyperglycaemia, and abdominal obesity [1]. Individuals with metabolic syndrome are at a significantly increased risk of developing chronic diseases such as type 2 diabetes and cardiovascular disease [1, 2]. Prevalence of metabolic syndrome varies between populations, with estimates of approximately 13-30 % of adults in developing countries and 35 % in developed countries such as Australia and the USA [3]. Data from the United Kingdom suggests that prevalence increases with age [3]. Several risk factors are responsible for the majority of metabolic syndrome cases and cardiovascular diseases in developed countries [4]. These include high cholesterol, overweight/obesity, physical inactivity, high blood pressure, and limited fruit and vegetable intake [4]. The risk factors contributing significantly to the burden of disease in Australia are poor diet (11 %) and overweight/obesity (9 %) [5]. In 2011–12, 92 % of Australian adults were not consuming enough serves of vegetables to meet the Australian Dietary Guidelines, and just 49 % achieved the recommended target for fruit consumption [5], while only 40 % met the recommendation of at least 150 min per week of moderate intensity physical activity [5]. The gap in health risk behaviours between the least and most disadvantaged groups of the Australian population is widening, particularly for fruit and vegetable intake [6]. People living in lower socioeconomic areas of Australia are more likely to be physically inactive, engage in sedentary behaviour, and have abnormal glucose metabolism compared to those living in higher socioeconomic areas [7]. Similar effects have been noted across seven comparable countries, with higher socioeconomic neighbourhoods associated with increased consumption of fruit and vegetables [8]. Residents of rural and remote Australia experience higher rates of morbidity and mortality and have less access to health services than those in metropolitan areas [5]. These populations are more likely than city populations to be overweight or obese (70 versus 60 %), insufficiently active (60 versus 54 %), have high blood cholesterol (37 versus 31 %) and comprise more people aged over 65 years (16 versus 13 %) [5]. This results in an increased prevalence of overweight/obesity, metabolic syndrome, and in turn chronic diseases [9]. Targeted screening and interventions in these populations at high risk of developing cardiovascular disease and type 2 diabetes may lead to early identification of metabolic syndrome, early management and delayed onset of these chronic diseases [10, 11]. Interventions to address metabolic syndrome and related chronic diseases in older adults should encourage reduced sitting time and increased physical activity [12] in combination with diet modifications. These include limiting saturated fat, sugar and salt, and increasing fibre, fruit and vegetable consumption [1, 13, 14]. The literature indicates that structured behavioural interventions focusing on counselling, education, and support strategies can also assist positive behaviour change in individuals at risk of chronic diseases [15]. A recent systematic review of self-help interventions for adults at risk of chronic diseases suggests that strategies comprising goal setting and self-monitoring in combination with tailored feedback, contact via email, and online social support, may be more effective for achieving behaviour change [16]. However, it is difficult to determine the effectiveness of self-help interventions in disadvantaged target groups due to insufficient data [16]. In particular, there is a gap in the knowledge of the impact of home-based lifestyle interventions in the disadvantaged rural/remote Australian setting targeting older adults with metabolic syndrome [17]. The Albany Physical Activity and Nutrition (APAN) Program aimed to improve dietary and physical activity behaviours of 50–69 year old adults at risk or with metabolic syndrome. The APAN program was implemented in the City of Albany and surrounding towns in The Great Southern region of Western Australia, which provided access to a large number of older adults aged 50–69 years (8496) [18]. The Socio-Economic Indexes for Areas scores Albany at 987.4, indicating relative disadvantage (<1000) [19]. This population is representative of other Australian rural/regional areas. In addition, the Great Southern region’s health profile suggests a need for increased access to health programs and services for residents [20]. The present study aimed to determine whether the APAN program effectively improved the physical activity, diet, and sedentary behaviours of participants. BODY.METHODS: BODY.STUDY DESIGN: The protocol of this trial has been described previously [21]. APAN was a two-arm randomised controlled trial of a 6-month physical activity, diet, and healthy weight management intervention. Data were collected from the intervention and control groups at baseline and post-test. The trial was registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12614000512628) and the study protocol was approved by the Curtin University Human Research Ethics Committee (approval number HR149_2013). All participants provided informed consent prior to entry into the study. BODY.PARTICIPANTS: Potential participants were required to be 50–69 years of age and classified as either at risk of, or with metabolic syndrome using the International Diabetes Federation (IDF) criteria [22] to be eligible for the study. To be considered at risk, participants were required to have a large waist circumference as the minimum requirement (waist circumference ≥94 cm for men or ≥80 cm for women [Europids, Sub-Saharan Africans, Eastern Mediterranean, Middle East]; ≥90 cm for men or ≥80 cm for women [South Asians, Chinese, Japanese]), plus one of the following parameters: raised triglyceride concentration (≥1.7 mM, or treatment for this); reduced HDL-cholesterol concentration (<1.03 mM in males and <1.29 mM in females, or treatment for this); raised blood pressure (systolic ≥130 mmHg or diastolic ≥85 mmHg, or treatment of previously diagnosed hypertension); raised fasting plasma glucose concentration (≥5.6 mM). Participants were classified as having metabolic syndrome if they had a large waist circumference plus two of the other criteria stated above [23]. The following exclusion criteria applied: on a weight loss diet or having weight fluctuations of >5 % within the previous 6 months; previous diagnosis of diabetes mellitus (other than gestational diabetes); of Aboriginal or Torres Strait Islander decent; receiving specific treatment to lower blood glucose; or involvement in another physical activity program. BODY.PROCEDURE: Participants aged 50–69 years were recruited from towns within a 50 km radius of Albany, Western Australia. Recruitment of participants occurred in three stages. Figure 1 outlines the study procedure, participant flow, and samples sizes.Fig. 1Consort flow diagram BODY.SCREENING STAGE 1: Individuals (n = 12,723) from the selected region were initially screened via the Computer Assisted Telephone Interview system using the Australian Type 2 Diabetes Risk Assessment Tool (AUSDRISK), which assesses the risk of developing type 2 diabetes mellitus based on anthropometric, demographic, and lifestyle characteristics [24]. Females with a score of ≥ 9 and males with a score of ≥ 12 were eligible for the next stage of screening (n = 1134), with 7342 individuals excluded and 4247 individuals who opted out. BODY.SCREENING STAGE 2: Eligible individuals (n = 1134) were invited to attend a clinic in a central location in Albany. A total of 526 participants attended the clinic and 608 opted out. Anthropometry including waist circumference was measured during the appointment to confirm central obesity (waist circumference ≥94 cm for men or ≥80 cm for women, being the minimum IDF requirement) before progressing to the next stage. Based on this eligibility criterion, 40 participants were excluded, and a further 16 opted out after progressing to screening stage 3. BODY.SCREENING STAGE 3: The remaining eligibility criteria were assessed at a local pathology laboratory via fasting blood sample and blood pressure measurements. Participants also completed a self-reported questionnaire to measure physical activity and dietary behaviours. Participants meeting the full eligibility criteria (n = 401) were randomly allocated to either the intervention group (n = 201) or control group (n = 200), while adjusting for gender and metabolic syndrome status. Intervention group participants were provided the APAN program materials and allocated to one of two motivational support staff. The control group participants were waitlisted to receive their program after post-test data collection (6 months). Baseline and post-test data collection included a self-reported questionnaire to measure participant dietary and physical activity behaviours. Of the 201 intervention and 200 control group participants who completed the questionnaire at baseline, 151 (75.1 % response rate) and 159 (79.5 % response rate) respectively completed the post-test questionnaire and were available for analysis of the self-reported outcome measures. BODY.INTERVENTION: This home-based program was low cost and tailorable to suit varying fitness levels and lifestyles. Its strategies included self-monitoring and goal setting, supported by printed and online material, and participants received email and telephone support using motivational interviewing techniques. The program built upon previous dietary and physical activity programs targeting older adults in a metropolitan area [25, 26], and was adapted for a rural/remote target group. BODY.THEORETICAL BASIS: The Self-Determination Theory was applied as the theoretical framework for this study, complemented by Motivational Interviewing [27]. The theory explores human motivation in terms of behaviours that are autonomous (originating from self) and behaviours that are controlled (persuasion or coercion) [28]. Self-Determination Theory is the only motivational theory that identifies autonomy as a basic need for all humans that should be supported in health promotion interventions [29]. Individuals are more likely to engage in certain behaviours if they are valued and intrinsically motivated [30]. Advice from health professionals should be offered in an autonomous manner to allow for individual decision-making [27]. BODY.PROGRAM MATERIALS: Intervention group participants were provided with the APAN program materials designed to assist with self-monitoring and goal setting to improve diet, physical activity, and healthy weight maintenance. Recommendations were based on Australia’s Physical Activity and Sedentary Behaviour Guidelines [31] and the Australian Dietary Guidelines [32], as well as previous successful interventions targeting older adults [25, 26]. Materials included a booklet, exercise charts, resistance band, nutrition panel wallet card, and a website with progress tracker and interactive blog. A detailed overview of the program materials has been described previously [21]. BODY.MOTIVATIONAL SUPPORT: Intervention group participants were provided motivational support for the duration of the APAN intervention via telephone counselling and emails. Two research assistants trained in motivational interviewing techniques contacted participants at week 1, 3, 6, 12, 18, and 24. Motivational interviewing principles incorporate strategies to encourage an individual to initiate and sustain behaviour change, such as empathy, shared decision-making, and reflective listening [33]. These strategies complemented the principles of the Self-Determination Theory by ensuring participants set their own goals, became involved in decision-making, and were not coerced into a particular behaviour or action [27, 30]. Research assistants were provided with a dialogue guide for each of the six MI telephone calls scheduled over the intervention period. The first call focused on general health as the main topic, allowing the research assistants to introduce themselves to participants and gauge their readiness to change, barriers to change, and identify specific goals they wished to achieve. Subsequent calls focused on specific topics including physical activity, sedentary behaviour, nutrition, and the revisiting of goals. Participants were encouraged to utilise their APAN resources to monitor their progress and adjust goals and behaviours as required. BODY.INSTRUMENT: The self-completed structured questionnaire included the Fat and Fibre Barometer [34], the International Physical Activity Questionnaire Short Form (IPAQ-SF) [35], and demographic and general health questions including sex, education, marital status, smoking status, diagnosed health conditions, medications, and alcohol consumption. The IPAQ-SF measured walking time, moderate and vigorous intensity physical activities, and time spent sitting across a usual week. A strength question was also added to determine resistance or weight training using large muscle groups [26]. To assess diet, the Fat and Fibre Barometer was utilised to provide information on habitual fat- and fibre-related behaviours of participants [34]. The valid and reliable instrument was appended with questions asking participants for the number of fruit and vegetable serves in a usual week [26]. Body mass index (BMI) was calculated based on weight and height data collected during screening stage 2. BODY.STATISTICAL ANALYSIS: This paper focuses on dietary and physical activity behaviours whereas changes in clinical metabolic syndrome parameters and anthropometry will be reported elsewhere. Descriptive statistics summarised the baseline lifestyle and demographic characteristics of the intervention and control groups. Independent and paired samples t-tests were applied to the continuous outcome variables, whereas Mann-Whitney U test and Wilcoxon Signed Rank test were applied to those variables exhibiting skewed distributions. To account for the effects of potential confounders, generalised estimating equation (GEE) models with exchangeable correlation structure were used to assess the repeated outcome variables over time. Normal GEE with identity link was applied to normally distributed continuous outcome variables (sitting time [hours per day]; fibre intake score; fat intake score; fat avoidance score), while gamma GEE with log link was applied to skewed continuous variables (walking time [MET min/week]; moderate intensity activity [MET min/week]; vigorous intensity activity [MET min/week]; total activity [MET min/week]; strength training [min/week]; fruit intake [serves per day]; vegetable intake [serves per day]). All statistical analyses were performed using the SPSS Statistics Package 22. BODY.RESULTS: Demographic and lifestyle characteristics of participants are summarised in Table 1, which highlight no significant differences between the intervention and control groups at baseline. The mean age of the sample was 61 years with 66.5 % female participants. The majority (98.4 %) had completed either secondary school or tertiary education, and 81.6 % had a partner. The mean BMI was 30.8 indicating that on average, the sample was obese. Smoking status and alcohol consumption were not significantly different between the two groups.Table 1Baseline characteristics of intervention and control group participants VariableIntervention groupControl group p valuea (n = 151)(n = 159)Age: mean (SD) years 60.5 (5.64) 61.3 (5.18) 0.181 Metabolic syndrome status: 0.494 With 79 (52.3 %) 90 (56.6 %) At risk 72 (47.7 %) 69 (43.4 %) BMI: mean (SD) 31.0 (5.88) 30.6 (5.17) 0.440 Gender: female 100 (66.2 %) 106 (66.7 %) 0.934 Employment status: 0.296 Full time 78 (51.7 %) 65 (40.9 %) Part time 24 (15.9 %) 29 (18.2 %) Unemployed 5 (3.3 %) 7 (4.4 %) Retired 44 (29.1 %) 58 (36.5 %) Education: 0.425 Primary school 3 (2.0 %) 2 (1.3 %) Secondary school 55 (36.4 %) 72 (45.0 %) Technical/Diploma 52 (34.4 %) 46 (28.8 %) University 41 (27.2 %) 39 (24.5 %) Relationship status: with partner 124 (82.1 %) 129 (81.1 %) 0.810 Smoking status: 0.852 Never 84 (55.6 %) 84 (52.8 %) Ex-smoker 52 (34.4 %) 54 (33.8 %) Occasional smoker 3 (2.0 %) 4 (2.5 %) Daily smoker 12 (7.9 %) 17 (10.6 %) Co-morbidityb: yes 92 (60.9 %) 104 (65.4 %) 0.413 Alcohol drinking: yes 99 (65.6 %) 113 (71.1 %) 0.957 a t-test or chi square test between intervention and control groups b Presence of at least one of 8 common health problems BODY.PHYSICAL ACTIVITY OUTCOMES: The self-reported physical activity outcomes between intervention and control groups are displayed in Table 2. Significant improvements in mean walking time (MET min/week), moderate intensity activity (MET min/week), total activity (MET min/week), sitting time (hours per day), and strength training (minutes per week) from baseline to post-test were observed for the intervention group, while a significant reduction (17 min/week) in total MET min/week was observed for the control group from baseline to post-test. Table 3 provides the results of the GEE analyses for physical activity outcomes. After controlling for potential confounders, the intervention group achieved a marginally significant improvement in the self-reported moderate intensity MET minutes per week (p = 0.049) relative to the control group. No significant improvement was observed for the other physical activity variables through the group x time interaction term.Table 2Comparison of self-reported physical activity outcomes between intervention and control groups OutcomeIntervention group (n = 151)p valuea Control group (n = 159) p valueb p valuec p valued BaselinePostBaselinePostMean (SD)Mean (SD)Mean (SD)Mean (SD)Walking MET min/weeke 396.0 577.5 0.011 330.0 396.0 0.047 0.561 0.050 (561.0) (742.5) (594.0) (709.0) Moderate MET min/weeke 300.0 480.0 <0.001 360.0 360.0 0.808 0.394 0.189 (585.0) (850.0) (640.0) (636.0) Vigorous MET min/weeke 181.5 217.5 0.070 203.0 179.5 0.715 0.902 0.176 (479.4) (460.2) (462.1) (428.4) Total MET min/weeke 807.5 1332.0 <0.001 990.0 973.0 0.013 0.428 0.051 (1486.9) (1624.9) (1357.5) (1738.0) Strength min/weeke 39.2 53.9 <0.001 27.7 25.0 0.436 0.880 0.001 (168.9) (124.2) (66.8) (70.4) Sitting hours/day 359.1 319.7 0.001 356.0 339.4 0.187 0.955 0.299 (187.4) (162.7) (171.3) (169.4) a Paired t-test (Wilcoxon signed-rank test) between baseline and post-test for the intervention group b Paired t-test (Wilcoxon signed-rank test) between baseline and post-test for the control group c Independent t-test (Mann-Whitney U test) between intervention and control group at baseline d Independent t-test (Mann-Whitney U test) between intervention and control group at post-test eNon-parametric tests applied Table 3Regression analysis of physical activity outcomes before and after intervention (n = 310) Group: interventionTime: postGroup x timeCoefficient (SE)pc Coefficient (SE)pc Coefficient (SE)pc Walking MET min/wka 0.09 (0.16) 0.580 0.23 (0.10) 0.020 −0.10 (0.16) 0.524 Moderate MET min/wka −0.07 (0.16) 0.650 0.01 (0.11) 0.991 0.29 (0.15) 0.049 Vigorous MET min/wka −0.21 (0.19) 0.275 −0.14 (0.17) 0.393 0.15 (0.24) 0.537 Total MET min/wka −0.05 (0.13) 0.710 0.12 (0.08) 0.164 0.11 (0.12) 0.335 Strength min/wka 0.01 (0.32) 0.983 −0.40 (0.22) 0.066 0.15 (0.34) 0.653 Sitting hours/dayb −10.61 (19.71) 0.590 −18.10 (12.54) 0.149 −20.86 (17.16) 0.224 aGamma generalised estimating equation model with log link bNormal generalised estimating equation model with identity link cAdjusted for age, gender, relationship status, education level, employment status, co-morbidity, alcohol drinking, and smoking status BODY.DIETARY OUTCOMES: The dietary outcomes are compared between groups in Table 4. Significant improvements in all self-reported outcome measures from baseline to post-test were observed for the intervention group, whereas the control group demonstrated no significant changes. Significant differences were observed between groups at post-test for the fibre intake score (p = 0.004), fat intake score (p < 0.001), fruit intake (p = 0.001), and vegetable intake (p < 0.001). Table 5 provides the results of the GEE analyses for dietary outcome variables. After controlling for potential confounders, the intervention group demonstrated significant improvements in the self-reported fibre intake (p < 0.001), fat intake (p = 0.003), and serves of vegetables per day (p = 0.002) using the group x time interaction term.Table 4Comparison of self-reported dietary outcomes between intervention and control groups OutcomeIntervention group (n = 151) p valuea Control group (n = 159) p valueb p valuec p valued BaselinePostBaselinePostMean (SD)Mean (SD)Mean (SD)Mean (SD)Fibre intake score 23.3 (4.2) 24.9 (4.1) <0.001 23.4 (4.0) 23.6 (3.8) 0.313 0.852 0.004 Fat avoidance score 12.8 (3.8) 13.3 (3.8) 0.016 12.8 (3.8) 12.8 (4.0) 0.743 0.933 0.234 Fat intake score 30.9 (4.0) 32.3 (4.0) <0.001 30.4 (3.8) 30.8 (3.4) 0.082 0.194 <0.001 Fruit intake (serves/day)e 1.5 (1.3) 2.0 (1.4) <0.001 1.5 (1.2) 1.4 (1.1) 0.378 0.619 0.001 Vegetable intake (serves/day)e 3.0 (2.3) 3.4 (1.9) <0.001 2.5 (2.2) 3.0 (2.3) 0.570 0.277 <0.001 a Paired t-test (Wilcoxon signed-rank test) between baseline and post-test for the intervention group b Paired t-test (Wilcoxon signed-rank test) between baseline and post-test for the control group c Independent t-test (Mann-Whitney U test) between intervention and control group at baseline d Independent t-test (Mann-Whitney U test) between intervention and control group at post-test eNon-parametric tests applied Table 5Regression analysis of dietary outcomes before and after intervention (n = 310) Group: interventionTime: postGroup x timeCoefficient (SE)pc Coefficient (SE)pc Coefficient (SE)pc Fibre intake scoreb −0.06 (0.45) 0.897 0.20 (0.20) 0.329 1.39 (0.32) <0.001 Fat intake scoreb 0.57 (0.43) 0.188 0.41 (0.23) 0.071 0.97 (0.33) 0.003 Fat avoidance scoreb 0.03 (0.42) 0.942 0.05 (0.18) 0.797 0.48 (0.28) 0.086 Fruit intakea 0.07 (0.07) 0.327 0.04 (0.04) 0.343 0.42 (0.26) 0.106 Vegetable intakea 0.07 (0.06) 0.292 0.04 (0.04) 0.321 0.17 (0.06) 0.002 aGamma generalised estimating equation model with log link bNormal generalised estimating equation model with identity link cAdjusted for age, gender, relationship status, education level, employment status, co-morbidity, alcohol drinking, and smoking status BODY.DISCUSSION: Poor diet, physical inactivity, sedentary behaviour, and overweight/obesity are some of the major risk factors contributing to Australia’s burden of disease [5]. These risk factors are more prevalent in disadvantaged and rural/remote communities in the older age groups [7], with the gap widening particularly for fruit and vegetable intake [6]. Consequently, interventions targeting this high risk group are essential to address the rising prevalence of obesity, metabolic syndrome, and chronic diseases in Australia [36, 37]. Assessment of changes to diet and physical activity behaviours of study participants provides an understanding of intervention compliance and the implication of changes in outcome measures [38]. This study examined the effectiveness of the APAN program for improving the dietary and physical activity behaviours of 50–69 year old adults at with, or at risk of metabolic syndrome in a disadvantaged rural area. The sample sizes provided sufficient statistical power to evaluate the repeated measures [21]. As expected, the attrition rate was higher for the intervention group (24.9 %) than the control group (20.5 %), and comparable to similar studies [39, 40]. The APAN program utilised the AUSDRISK [24] to initially screen individuals for risk of metabolic syndrome based on their risk of developing type 2 diabetes. To date no other studies report using this screening tool to identify metabolic syndrome in large populations. The screening process identified 1060 high risk individuals for developing type 2 diabetes, of which 215 (20 %) were confirmed to have metabolic syndrome and 186 (18 %) at risk of metabolic syndrome using the IDF criteria (central obesity plus one instead of two of the additional parameters). Targeted screening using AUSDRISK and the IDF metabolic syndrome criteria allows for evaluation of a timely lifestyle intervention to prevent the onset of cardiovascular disease and type 2 diabetes in rural Australian communities. It is recommended that lifestyle interventions for disadvantaged groups incorporate self-help strategies due to the potentially high reach and low cost of implementation [16]. A systematic review of optimal methods and strategies for achieving lifestyle behaviour change in individuals with metabolic syndrome reported that interventions incorporating motivational feedback/interviewing in combination with internet monitoring and regular personal feedback are likely to achieve the best results [41]. Additionally, telephone-based services are able to reach geographically and socially disadvantaged areas, which commonly have higher risk of chronic diseases [42]. The APAN program incorporated a combination of these strategies to ensure the program was delivered in a cost-effective manner to the relatively disadvantaged participants in a rural community [19]. The observed changes to diet (fat, fibre and vegetable intake) and physical activity behaviours (moderate intensity activity) for the intervention group suggest that this combination of strategies is effective for the high-risk target group. The APAN program was based on the Australian Dietary Guidelines [32]. Participants were encouraged to consume a diet high in fruit and vegetables with an emphasis on fibre intake, which is the recommendation for individuals with or at risk of metabolic syndrome [43]. The significant improvement in vegetable consumption and fat and fibre intake for the intervention group suggests that the APAN program demonstrates protective dietary factors to address metabolic syndrome in the target group. The APAN program provided strategies for participants to identify and overcome barriers to improving physical activity and diet. Identified barriers to fruit and vegetable intake in the literature include a perception that enough serves of fruit and vegetables were consumed, as well as lack of variety, difficulty changing habits, lack of time to prepare, and low quality produce [44]. It has been suggested that interventions should provide practical solutions to address these barriers, and devise strategies to effectively communicate the recommended serves of fruit and vegetables to participants [44]. The APAN program supported participants by providing detailed information on food groups, recommended serves per day, sample meal plans, and tips for incorporating more fruit and vegetables into their daily meals, and the motivational interviewing component of the intervention encouraged participants to utilise these tools on a regular basis. Therefore, the significant increase in serves of vegetables per day for the intervention group is a successful achievement. Based on Australia’s Physical Activity and Sedentary Behaviour Guidelines [31], the APAN program recommended that participants aim for at least 150 min per week of moderate intensity physical activity in combination with a reduction in sitting time and strength training, as these activities all impact on metabolic syndrome outcomes. There was a marginal improvement in moderate intensity activity, which is encouraging as the risk of metabolic syndrome is almost doubled among adults who engage in no moderate intensity activity [43]. Although the intervention group demonstrated some improvement in sitting time, this change was not significantly different relative to the controls. Further research into strategies to encourage a reduction in sitting time in the home environment is recommended for the target group, including optimal motivational interviewing techniques to achieve this. Loss of muscle mass and strength, or sarcopenia, affects wellbeing, physical movement, glycaemic control and blood pressure [45, 46]. Strength training is therefore a crucial component of any physical activity intervention targeting older adults due to its physiological effects [46]. The prevalence of strength training in Australian adults remains low, particularly in regional settings (21.9 % for men and 17.5 % for women) and adults aged 55+ (7 %) [46]. The intervention group did not achieve a significant increase in strength training time compared to the control group, highlighting the need for more effort to be placed on this. The APAN exercise chart was adapted from a previous study targeting older adults (60+ years) which might have been too simplistic and not challenging enough for the younger target group in the current study. Further adaptations should be considered for future interventions. BODY.LIMITATIONS: A limitation of this study is the use of self-reported outcome measures which introduces reporting bias; however, any inaccuracies in reporting are anticipated to be the same across intervention and control groups. Additionally, allocation concealment ensured that the participants were unaware of their group allocation which reduced the effect of any differential over-reporting of desired behaviours and under-reporting of undesired behaviours. Another limitation is the short duration of the intervention (6 months). It is commonly accepted that exposing older adults to a sufficient dose of an intervention to ensure behaviour change is maintained in the long term can be challenging [47]. Risk factors for metabolic syndrome are effectively controlled by intensive, short-term programs for weight loss; however once the programs end there are high rates of recidivism and individuals have a tendency to regain weight [41]. A follow-up study is therefore recommended to determine long-term effectiveness and sustainability of the APAN program. BODY.CONCLUSIONS: Rural and remote communities are often neglected in lifestyle intervention research. It is therefore imperative that future interventions focus on sustainable health outcomes in real-world settings, particularly in disadvantaged groups. Considering the increased prevalence of preventable disease risk factors, it is essential to address poor diet, physical inactivity, and sedentary behaviour to reduce the prevalence of metabolic syndrome and related chronic diseases. The APAN program improved the physical activity and diet of the intervention group compared to the control group, demonstrating that a home-based, low-cost intervention with motivational support can effectively influence certain behaviour change of 50–69 year old adults with or at risk of metabolic syndrome in a disadvantaged rural area. The study findings thus contribute to the prevention and control of chronic diseases.

TITLE: Environmental temperature and exercise modality independently impact central and muscle fatigue among people with multiple sclerosis ABSTRACT.BACKGROUND: Heat sensitivity and fatigue limit the ability of multiple sclerosis patients to participate in exercise. ABSTRACT.OBJECTIVE: The purpose of this study was to determine the optimal aerobic exercise parameters (environmental temperature and exercise modality) to limit exercise-induced central and muscle fatigue among people with multiple sclerosis. ABSTRACT.METHODS: Fourteen people with multiple sclerosis with varying levels of disability completed four randomized exercise sessions at 65% of the maximal volume of oxygen: body-weight supported treadmill cool (16°C), body-weight supported treadmill room (21°C), total-body recumbent stepper cool and total-body recumbent stepper room. Maximum voluntary contraction, electromyography, and evoked contractile properties were collected from the more affected plantar flexors along with subjective levels of fatigue, body temperature and perceived level of exertion. ABSTRACT.RESULTS: Exercise in cooler room temperature increased maximum voluntary contraction force (p = 0.010) and stabilized body temperature (p = 0.011) compared to standard room temperature. People with multiple sclerosis experienced greater peak twitch torque (p = 0.047), shorter time to peak twitch (p = 0.035) and a longer half relaxation time (p = 0.046) after total-body recumbent stepper suggestive of less muscle fatigue. ABSTRACT.CONCLUSION: Cooling the exercise environment limits the negative effects of central fatigue during aerobic exercise and using total-body recumbent stepper (work distributed among four limbs) rather than body-weight supported treadmill lessens muscular fatigue. Therapists can titrate these two variables to help people with multiple sclerosis achieve sufficient exercise workloads. BODY.INTRODUCTION: Exercise training improves muscular strength, aerobic capacity, walking ability and health-related quality of life among people with multiple sclerosis (PwMS).1 However, due to disability, fatigue and heat sensitivity, most PwMS do not exercise2,3 and so they do not obtain its potential benefits. Aerobic fitness, in particular, is associated with less brain atrophy and better cognitive function in multiple sclerosis (MS)4 but at the same time, aerobic exercise increases body temperature which many MS patients find intolerable.5 For example, after leg cycling, MS participants experienced greater increases in body temperature compared to controls which was correlated with prolonged perceived leg fatigue.6 There is a need to find innovative and tailored exercise protocols so that PwMS can achieve optimal exercise workloads.7 Exercise-induced fatigue could be due to impaired motor drive from the central nervous system (CNS) (central fatigue) or reduced capacity from within the muscle.8,9 Research supports the interplay of both mechanisms. Demyelination of motor pathways,10 impaired thermoregulation, and transient increase in symptoms with passive heat exposure5 point to a central origin. However, decreased muscle peak twitch (PT) force output and prolonged half-relaxation time (HRT) during fatiguing electrical stimulation of the tibialis anterior in PwMS11 is suggestive of peripherally induced fatigue within the muscle.11,12 Aerobic exercise is typically performed on a treadmill but can be adapted for PwMS who have balance difficulties by using seated or recumbent methods. For example, Pilutti et al.13 compared the safety and tolerability of the total body recumbent stepper (TBRS) to the body-weight supported treadmill (BWST) and found that 12 weeks of TBRS and BWST training reduced perceived fatigue, although PwMS reported a more positive experience with the TBRS.13 It is conceivable that distributing the workload between four limbs rather than two could lessen excessive lower limb fatigue. Choosing the appropriate exercise modality (seated or upright) and cooling the temperature of the exercise environment are simple methods that therapists and patients can employ in order to titrate aerobic exercise parameters to make training achievable. The primary aim of this study was to determine the acute effects of combining temperature ('cool', 16°C or 'room', 21°C) and exercise modality (BWST or TBRS) on lower extremity central fatigue (maximum voluntary contraction (MVC)), muscle fatigue (evoked contractile properties), body temperature, and perceived levels of fatigue. We hypothesized that: (a) exercising in a cooler temperature would prevent post-exercise decreases in MVC and electromyography (EMG) and (b) due to distributed workload between all four limbs, PwMS would have less perceived fatigue following the TBRS compared with the BWST. In order to understand potential mechanisms, our exploratory aims were to evaluate the relationships between exercise-induced changes in body temperature, subjective fatigue, the extent of clinical disability, and changes in neuromuscular performance. BODY.METHODS: Following approval of the Health Research Ethics Board (ref. no. 14.102), participants were recruited from outpatient rehabilitation services and the local MS clinic. From these participants, 14 PwMS (10 females) were included; they all met the following inclusion criteria: (a) diagnosis using the McDonald criteria;14 (b) a negative Physical Activity Readiness Questionnaire (PAR-Q)15 screening; (c) being relapse-free during the past three months; (d) taking no medication that affects heart response to exercise; (e) having no musculoskeletal impediment to exercise; and (f) scoring greater than 24 on the Montreal Cognitive Assessment (MoCA).16 BODY.EXPERIMENTAL DESIGN: During the first session, demographic and MS-related data were collected (age, years since diagnosis, Fatigue Impact Scale (FIS)).17 PwMS self-reported their degree of heat sensitivity using a visual analogue scale (VAS); zero being not at all sensitive and 100 being extremely sensitive. Lower limb strength was tested using manual muscle testing, spasticity using the Modified Ashworth Scale and self-selected walking velocity using an instrumented walkway (Protokinetics, Havertown, Pennsylvania, USA). BWST and TBRS graded maximal exercise tests (GXTs) were performed (Figure 1(a)) and maximal volume of oxygen (V̇O2max) was used to assign 60–65% of heart rate (HR) reserve for 30 min of aerobic exercise (including a five-minute warm-up and cool down). The participants completed four randomized exercise sessions, one week apart either on TBRS or BWST in a temperature controlled room at 16°C (cool) or 21°C (room); BWSTcool, BWSTroom, TBRScool, TBRSroom (Figure 1(a)). Rate of perceived exertion (RPE; Borg Scale (10-point)) and HR were recorded at intervals 1, 15, and 30 min during exercise. Prior to each exercise session, participants were prepared for EMG (Figure 1(b)) and performed isometric contractions of the plantar flexors at various low intensities as a warm-up. Before and after exercise, participants performed two MVCs, received posterior tibial nerve stimulation, rated their perceived fatigue on a 100 mm VAS, and had body temperature recorded using a tympanic thermometer (Thermoscan, Braun, Kronberg, Germany). Figure 1.(a) Experimental design. Graded maximal exercise tests (GXTs) were performed one week apart. Sessions 3–6 were performed in randomized order one week apart; body-weight support treadmill at room 21°C (BWSTroom); BWST at 16°C (BWSTcool); total-body recumbent stepper at 21°C (TBRSroom); TBRS at 16°C (TBRScool). (b) Intervention session protocol: before and after exercise, participants performed two maximal voluntary contractions (MVCs) and received posterior tibial nerve electrical stimulation to measure plantar flexor twitch contractile properties from lateral gastrocnemius (LG) and soleus (SOL) muscles. VO2max: maximal volume of oxygen. BODY.MAXIMAL EXERCISE TESTS: V̇O2max test was performed on both the BWST using 10% body weight support (Sport Art T625M/T52 MD-Rehabilitation Commercial Treadmill, USA) and the TBRS (NuSTEP T4r Recumbent stepper, Michigan, USA), according to guidelines adapted from stroke best practices.18 Height, weight, age, resting HR, and blood pressure were taken prior to the GXT. The metabolic cart (Moxus Metabolic Systems, AEI Technologies, Inc., Pittsburgh, Pennsylvania, USA) was calibrated and expired air was analyzed breath-by-breath using a mask (HR: Polar V800, Polar Electro Oy, Professorintie 5, FI-90440 Kempele, Finland). BODY.MEASUREMENT OF PLANTAR FLEXOR MUSCLE FORCE AND ELECTROMYOGRAPHY: Participants sat with their weaker leg flexed (i.e. 90° at the hip, knee, and ankle joints) and foot mounted in a modified boot apparatus equipped with a load cell, and performed two five-second MVCs. Participants were told to push into plantar flexion as hard as possible for five seconds. Force was sampled at 1 kHz, amplified (×1000) and averaged. EMG was recorded via surface 10 mm electrodes (MediTrace Pellet Ag/AgCl, Graphic Controls Ltd., Buffalo, New York, USA) placed longitudinally 2 cm apart over the lateral gastrocnemius (LG) and soleus (SOL) muscles of the weaker leg (determined by manual muscle test) and a ground electrode was secured on the lateral epicondyle of the femur. Signals were amplified (1000×), filtered using a Butterworth filter with a pass-band of 10–500 Hz and analog-digitally converted at a sampling rate of 1000 Hz (Biopac MP150WSW, Biopac Systems Inc., Holliston, Mississippi, USA). Data were recorded and analyzed (Acknowledge 4.1, Biopac Systems Inc.). MVC forces were measured as the peak force recorded during each contraction. For the LG and SOL muscles, root mean square (RMS) EMG was calculated over 500 ms about the peak force amplitude during the MVC for each muscle. BODY.ASSESSMENT OF EVOKED CONTRACTILE PROPERTIES: To assess twitch contractile properties in LG and SOL muscles; electrical stimulation was applied to the posterior tibial nerve via Ag/AgCl electrodes placed in the popliteal fossa (cathode) and over the tibial tuberosity (anode). Current pulses (200 µs duration, 100–400 mA) were delivered via a constant current stimulator (DS7AH; Digitimer, Welwyn Garden City, Hertfordshire, UK). Stimulation intensity was increased until PT torque of LG and SOL plateaued. Evoked contractile properties included: (a) PT torque, (b) time to peak twitch (TPT), and (c) HRT – the time it took for the PT torque to reduce to half of its maximum amplitude. Lower PT and longer TPT are indicative of fatigue and lower efficiency of fiber cross-bridge cycling, respectively.19 BODY.STATISTICAL ANALYSIS: To determine whether the exercise conditions were at the equivalent aerobic intensity, repeated measures analysis of variance (ANOVA) for exercise condition (TBRS, BWST) and temperature (cool, room) were performed on HR, power output, and RPE averaged across three time points (1 min, 15 min, 30 min) during the 30 min of aerobic exercise. A repeated measures ANOVA for exercise condition (TBRS, BWST) by temperature (cool, room) was also performed on change scores (CSs) (post minus pre; MVCcs, RMS EMGcs, PTcs, TPTcs, HRTcs, fatiguecs, body tempcs,) and on perceived level of fatigue. Based on evidence that body temperature relates to changes in central drive in PwMS following exercise,20 correlations were performed between post-exercise MVC torque, degree of heat sensitivity and change in body temperature for each exercise modality (BWST; TBRS) in the room temperature condition. In exploratory analyses of MS biomarkers, the relationship between disability (Expanded Disability Status Scale (EDSS)) and variables at baseline (i.e. the average of pre-MVC, RMS EMG, PT, TPT, HRT, and perceived fatigue) was tested using Pearson correlations (Pearsons’s r: p < 0.05, uncorrected). If significant main interactions were found (F-ratio: p < 0.05), a Bonferroni post-hoc test was performed. If the assumption of sphericity was violated, the corrected value for non-sphericity with Greenhouse-Geisser epsilon was reported. Effect sizes were reported as partial eta-squared (np2) where 0.02 is considered a small effect, 0.13 moderate and more than 0.26, a large effect.21 Data are reported as means and standard errors (SEs) (SPSS 19.0, IBM Corporation, Armonk, New York, USA). BODY.RESULTS: Three of the 14 individuals were unable to complete the BWST protocol, and therefore were excluded from repeated measures statistical analyses (n = 11). All participants reported heat sensitivity (Figure 2) and 11 had lower extremity weakness (Table 1). Participant characteristics are reported in Table 1 and raw data for voluntary and evoked contractile properties are presented in Table 2. Figure 2.Ratings of heat sensitivity rating on heat sensitivity visual analog scale (VAS; score out of 100). Each circle is a participant. Dashed line is the mean and horizontal error bar is the standard deviation (SD). Table 1.Participants’ clinical characteristics. ParticipantEDSSAgeSexType of MSFISLE summed strengthLE spasticityUse of gait aidWalking speed cm/s1 2.0 31 Male RRMS 16 80 – – 128.19 2 1.5 38 Male RRMS 41 61 – + 79.15 3 4 56 Female RRMS 20 74 – – 97.29 4 3.5 52 Male RRMS 49 61 – – 84.37 5 1.5 65 Female RRMS 3 74 – + 110.86 6 7 55 Female SPMS 41 47 + + 32.16 7 1.5 43 Female RRMS 12 80 – – 119.75 8 6.5 71 Female SPMS 30 50 + + 16.45 9 1 28 Female RRMS 2 78 – – 123.54 10 6.5 59 Female PPMS 26 57 – + 46.06 11 0 29 Female RRMS 0 80 – – 76.50 12 2 60 Male RRMS 41 80 – – 119.22 13 0 53 Female RRMS 31 61 – – 143.20 14 1 50 Female RRMS 36 66 – – 124.13 EDSS: Expanded Disability Status Scale; FIS: Fatigue Impact Scale; LE: lower extremity; MS: multiple sclerosis; PPMS: primary progressive MS; RRMS: relapsing–remitting MS; SPMS: secondary progressive MS. Strength/80 summed manual muscle tests of major muscles in both lower limbs. Present (+), absent (–); walking speed, self-selected pace (cm/s). Table 2.Raw data for voluntary and evoked contractile properties. MeasureExerciseTempPre- (n = 11)Post- (n = 11) MVC (Nm) BWST Cool 89.81 (± 62.41) 100.3 (± 66.83) MVC (Nm) TBRS Cool 95.18 (± 53.19) 108.9 (± 61.89) MVC (Nm) BWST Room 112.3 (± 68.66) 101.1 (± 55.72) MVC (Nm) TBRS Room 98.90 (± 62.72) 93.68 (± 64.79) LG EMG (RMS) BWST Cool 0.063 (± 0.0359) 0.065 (± 0.0402) LG EMG (RMS) TBRS Cool 0.062 (± 0.0334) 0.072 (± 0.0488) LG EMG (RMS) BWST Room 0.076 (± 0.0446) 0.062 (± 0.0404) LG EMG (RMS) TBRS Room 0.082 (± 0.0582) 0.072 (± 0.0593) HRT (ms) BWST Cool 121.2 (±25.96) 110.3 (±21.22) HRT (ms) TBRS Cool 117.9 (±22.37) 104.6 (±27.80) HRT (ms) BWST Room 132.8 (±34.50) 108.1 (±28.23) HRT (ms) TBRS Room 107.4 (±26.94) 108.9 (±24.20) PT (Nm) BWST Cool 15.09 (± 9.644) 13.76 (± 7.101) PT (Nm) TBRS Cool 14.09 (± 6.245) 16.76 (± 10.12) PT (Nm) BWST Room 15.07 (± 6.702) 14.99 (± 8.750) PT (Nm) TBRS Room 14.46 (± 6.835) 16.75 (± 9.719) TPT (ms) BWST Cool 0.15 (± 0.0213) 0.14 (± 0.0196) TPT (ms) TBRS Cool 0.16 (± 0.0255) 0.15 (± 0.0347) TPT (ms) BWST Room 0.13 (± 0.0504) 0.14 (± 0.0255) TPT (ms) TBRS Room 0.17 (± 0.0229) 0.14 (± 0.0274) Maximal Voluntary Contraction (MVC); Lateral Gastrocnemius (LG); Electromyography (EMG); Newton Meter (Nm); Root Mean Square (RMS); Temperature (Temp); Half Relaxation Time (HRT); Peak Twitch (PT); Time to Peak Twitch (TPT); milliseconds (ms); Total body recumbent stepper (TBRS); Body-weight supported treadmill (BWST). BODY.THE INTENSITY OF EXERCISE WAS EQUIVALENT BETWEEN EXERCISE MODALITIES: There was no significant main effect of environmental temperature or exercise modality on HRavg or power outputavg, (data not shown). Although there was no significant main effect of environmental temperature on RPEavg (F(1,11) = 1.00, p = 0.34, np2 = 0.091), there was a significant main effect of exercise modality, (F(1,11) = 8.07, p = 0.018, np2 = 0.447) with a higher level of perceived exertion during TBRS (4.54 ± 0.278) compared to BWST (3.97 ± 0.293). There was no significant interaction of temperature and exercise modality (p = 0.17). BODY.MAXIMAL PLANTAR FLEXOR ACTIVATION WAS TEMPERATURE-DEPENDENT: There was a significant main effect of environmental temperature on MVCcs (Figure 3; F(1,11) = 9.86, p = 0.010, np2 = 0.497). MVC torque increased post-exercise in cool temperature (12.13 ± 4.54 Nm) and decreased in room temperature (–8.20 ± 5.13 Nm). There was a near main effect of temperature (F(1,11) = 6.73, p = 0.057, np2 = 0.254) but no effect of exercise modality on LG EMGcs. LG EMG remained the same post-exercise in cool temperature (0.002 ± 0.003 mV) but decreased in room temperature (–0.01 ± 0.004 mV). Figure 3.Temperature effects on maximal voluntary contraction (MVC). Change in MVC torque in room (black) and cool (black/white) collapsed across exercise modalities. *Indicates a significant (p<0.05) main effect of temperature condition. There was no effect of exercise modality on MVCcs, LG EMG or SOL EMG and no significant interaction between temperature and exercise modality. There was also no effect of temperature on SOL EMGCS (data not shown). BODY.EXERCISE MODALITY AFFECTED EVOKED CONTRACTILE PROPERTIES: Following BWST there was evidence of plantar flexor fatigue. There was a significant main effect of exercise modality on plantar flexor PTcs, (Figure 4; F(1,11) = 5.11, p = 0.047, np2 = 0.338) with decreased PT amplitude following BWST (–0.71 ± 0.982 Nm) and an increase following TBRS (2.48 ± 1.173 Nm). There was also a significant main effect of exercise modality on TPTcs (F(1,11) = 5.92, p = 0.035, np2 = 0.372) with prolonged TPT following BWST (0.004 ± 0.009 ms) and shortened TPT following TBRS (–0.019 ± 0.007 ms). Further, there was a significant main effect of exercise modality on HRTcs, (F(1,11) = 5.21, p = 0.046, np2 = 0.343) with shortened HRT following exercise on a BWST (–17.80 ± 2.845 ms) compared to exercise on TBRS (–5.89 ± 4.487 ms). Figure 4. Exercise modality effects on evoked contractile properties. Change in peak twitch (PT) in body-weight support treadmill (BWST) (black/white circle) and total-body recumbent stepper (TBRS) (black circle) collapsed across temperature conditions. *Indicates a significant (p<0.05) main effect of exercise modality. There was no effect of temperature on PTcs, TPTcs or HRTcs, and no significant interaction between temperature and exercise modality (data not shown). BODY.EFFECTS OF EXERCISE MODALITY ON PERCEIVED LEVEL OF FATIGUE AND BODY TEMPERATURE: There were significant main effects of exercise modality and environmental temperature on body tempcs (Table 3; F(1,9) = 7.61, p = 0.022, np2 = 0.458, F(1,9) = 10.08, p = 0.011, np2 = 0.528, respectively). Greater increases in body temperature were observed following exercise in room temperature (0.33 ± 0.104°C) compared to cool temperature (–0.15 ± 0.123°C). There was a greater increase in body temperature following TBRS (0.29 ± 0.094°C) compared to BWST (0.025 ± 0.126°C). There was no significant main effect of exercise modality or environmental temperature on perceived level of fatiguecs and no significant interaction of exercise modality and temperature (data not shown). Table 3.Raw data for change in body temperature (data are reported as mean (M) ± standard deviation (SD)) MeasureExerciseTempPre-(n = 11)Post-(n = 11)Body temp (°C) BWST Cool 36.65 (±0.454) 36.45 (±0.780) Body temp (°C) TBRS Cool 36.65 (±0.418) 36.74 (±0.559) Body temp (°C) BWST Room 36.49 (±0.287) 36.73 (±0.402) Body temp (°C) TBRS Room 36.35 (±0.462 36.78 (±0.524) BWST: Body-weight supported treadmill; TBRS: Total body recumbent stepper; Temp: temperature. BODY.RELATIONSHIP BETWEEN BODY TEMPERATURE, DEGREE OF HEAT SENSITIVITY AND NEUROMUSCULAR PERFORMANCE: Since there was little change in body temperature in the cool condition, changes in body temperature in the room temperature conditions were compared to the corresponding post-exercise MVC torque. Higher body temperature was correlated with declines in post-exercise MVC torque in BWST (Figure 5; r = 0.65, p = 0.028) and a nearly significant negative relationship in TBRS (r = 0.58, p = 0.075). There was no relationship between body temperature and evoked contractile properties. Nether was there a relationship between perceived fatigue, degree of heat sensitivity, MVC, EMG, or evoked contractile properties. Figure 5. Relationship between body temperature and post-exercise maximal voluntary contraction (MVC). Following body-weight support treadmill at room 21°C (BWSTroom),lower MVC torque was significantly correlated with increases in body temperature. *Indicates a significant (p < 0.05) correlation. BODY.RELATIONSHIP BETWEEN MS-RELATED DISABILITY AND NEUROMUSCULAR PERFORMANCE: As expected, higher EDSS scores were significantly correlated with lower MVC (r = –0.54, p = 0.045), lower LG (r = 0.62, p = 0.022), SOL RMS EMG (r = –0.66, p = 0.013), and greater baseline perceived fatigue (r = 0.67, p = 0.012). BODY.DISCUSSION: The main findings of the current study were (a) with large effect sizes, a cooler environment (16°C) limited elevation of body temperature and increased plantar flexor MVC torque and protected against decrements in EMG after exercise, suggesting an effect of temperature on CNS drive and (b) exercising on a recumbent stepper using all four limbs enhanced plantar flexor contractile properties as compared to treadmill training, suggesting an effect of exercise modality on the excitation-contraction coupling of the muscle. These findings occurred despite the fact that the exercise interventions were matched for workload and participants perceived the exercise on the TBRS as more strenuous than the BWST. Finally, the neuromuscular performance measures, specifically MVC and RMS EMG of the lower limb, correlated with clinical disability (EDSS), strengthening their usefulness as biomarkers of subtle MS-related sensorimotor change. BODY.ENVIRONMENTAL TEMPERATURE AND CENTRAL DRIVE: In the present study, exercising in room temperature (raising body temperature) led to decreased MVC torque and LG EMG, with no concomitant changes in evoked contractile properties post-exercise, pointing to CNS mechanisms underlying these changes. Other studies support such a theory. When PwMS were exposed to passive body heating, walking, force generation capacity, and increased fatigue perception were affected.22 Furthermore, passively-heated PwMS had decreased corticospinal excitability as shown via increased resting motor threshold and decreased motor evoked amplitude.22 Increased body temperature may also alter action potential propagation in demyelinated or partially myelinated axons causing reduced conduction velocity and/or block.5 In the present study, exercising in cool temperature enhanced MVC torque with no change in LG EMG. The cool temperature may have alleviated heat-induced stress on the CNS, allowing for improved MVC performance. Exercising in warm or cool environments, and rate of heat storage in the body, have been postulated to differentially activate an anticipatory response that regulates skeletal muscle power output and motor unit recruitment through afferent sensory inputs to the brain.23 Other studies have shown that, in PwMS, water immersion precooling before arm-leg ergometry exercise stabilized core temperature and improved 25-foot walk performance24 and that the use of cooling suits improved walking speed and lower-limb strength.25 Decreasing body heat storage, especially during exercise, may allow for improved CNS function and enhanced neuromuscular performance. Since comfort is likely important for exercise compliance, the longer-term effects (over hours and days) of temperature and fatigue on neuromuscular measures and functional tasks is an important area for future study. BODY.EXERCISE MODALITY: Based on our findings that BWST resulted in lower PT force and longer TPT, the BWST may create impaired excitation-contraction coupling of the plantar flexor muscles, indicating greater muscle fatigue, compared to the TBRS. Conversely, the TBRS facilitated the excitation-contraction coupling of the plantar flexor muscles demonstrated by increased PT force and shorter TPT. The enhancement of plantar flexor muscle contractile properties following the TBRS may be related to exercise-induced post-activation potentiation (PAP); enhanced calcium kinetics, myosin phosphorylation, and reduced muscle stiffness after non-fatiguing contractions.26 Biomechanically, the distributed workload between the upper and lower limb muscles required to perform the TBRS at a comparable physiological workload as submaximal BWST exercise, that predominately engages muscles of the lower limb, may help to explain our findings. For those with fatigue and heat sensitivity, choosing the TBRS in a cooler environment may allow patients to reach optimal aerobic exercise training levels. BODY.PERCEPTIONS OF FATIGUE: In a clinical setting, the patient’s subjective report of heat sensitivity and fatigue during exercise may be used when titrating aerobic training intensity. However, in this study, perceived fatigue following exercise was not related to objective measures of central or muscular fatigue, illustrating a disconnect between psychological vs physiological aspects of fatigue in PwMS following exercise. This disconnect suggests that subjective reports may not be sensitive enough to gauge heat-related fatigue. Dawes et al.20 showed that PwMS reporting general fatigue could train at comparable levels to those without fatigue suggesting that clinicians, in addition to obtaining subjective reports, should focus on objective markers of exercise intensity such as HR to gauge exercise parameters. Our findings suggest that, at least within a training session, the TBRS may be more tolerable especially for those with higher levels of MS-related disability. However, due to the importance of the exercise-induced stress response that is incurred from challenging exercise,27 transition to the BWST may be warranted as training progresses. BODY.DETECTING SUBCLINICAL CHANGES IN NEUROMUSCULAR PERFORMANCE: We found not only that there was an inverse relationship between EDSS score and MVC force and EMG of the plantar flexor muscles, but also the neuromuscular measures detected subtle deficits sometimes unbeknownst to the participant. Others have demonstrated that MVC, central activation,28 and evoked contractile properties10 are decreased in PwMS compared to controls and there is a negative correlation between EDSS and measures of corticospinal excitability.29 In a disease where most of the degeneration is subclinical and where lesions can outnumber clinical relapses 10 to one,30 there are opportunities to incorporate neuromuscular performance outcomes as potential biomarkers of MS disease progression. BODY.STUDY LIMITATIONS: This study makes important strides in developing tailored aerobic exercise interventions for PwMS. However, there were some limitations. Even though effect sizes were moderate to large, the sample size was small and because of inability to complete the BWST, participants with EDSS >6 were underrepresented. Body temperature was measured using the tympanic method. The most sensitive methods to measure thermoregulation and changes in core temperature are invasive, and are recorded using rectal and esophageal techniques. However, the aim of the present study was to use methods that could be easily incorporated into clinical practice and inform best practice guidelines for clinical exercise prescription. BODY.CONCLUSIONS: Cooling the temperature of the training environment lessens central fatigue while choosing a modality that distributes workload among four limbs (TBRS) rather than two (BWST) limits muscular fatigue. Therapists can modify these parameters in order to achieve optimal aerobic exercise training levels among PwMS.

TITLE: Effect of a Community-Based Nursing Intervention on Mortality in Chronically Ill Older Adults: A Randomized Controlled TrialCommunity Nursing and Mortality in Chronically Ill Kenneth Coburn and colleagues report findings from a randomized trial evaluating the effects of a complex nursing intervention on mortality risk among older individuals diagnosed with chronic health conditions. ABSTRACT.BACKGROUND: Improving the health of chronically ill older adults is a major challenge facing modern health care systems. A community-based nursing intervention developed by Health Quality Partners (HQP) was one of 15 different models of care coordination tested in randomized controlled trials within the Medicare Coordinated Care Demonstration (MCCD), a national US study. Evaluation of the HQP program began in 2002. The study reported here was designed to evaluate the survival impact of the HQP program versus usual care up to five years post-enrollment. ABSTRACT.METHODS AND FINDINGS: HQP enrolled 1,736 adults aged 65 and over, with one or more eligible chronic conditions (coronary artery disease, heart failure, diabetes, asthma, hypertension, or hyperlipidemia) during the first six years of the study. The intervention group (n = 873) was offered a comprehensive, integrated, and tightly managed system of care coordination, disease management, and preventive services provided by community-based nurse care managers working collaboratively with primary care providers. The control group (n = 863) received usual care. Overall, a 25% lower relative risk of death (hazard ratio [HR] 0.75 [95% CI 0.57–1.00], p = 0.047) was observed among intervention participants with 86 (9.9%) deaths in the intervention group and 111 (12.9%) deaths in the control group during a mean follow-up of 4.2 years. When covariates for sex, age group, primary diagnosis, perceived health, number of medications taken, hospital stays in the past 6 months, and tobacco use were included, the adjusted HR was 0.73 (95% CI 0.55–0.98, p = 0.033). Subgroup analyses did not demonstrate statistically significant interaction effects for any subgroup. No suspected program-related adverse events were identified. ABSTRACT.CONCLUSIONS: The HQP model of community-based nurse care management appeared to reduce all-cause mortality in chronically ill older adults. Limitations of the study are that few low-income and non-white individuals were enrolled and implementation was in a single geographic region of the US. Additional research to confirm these findings and determine the model's scalability and generalizability is warranted. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT01071967 Please see later in the article for the Editors' Summary ABSTRACT.EDITORS' SUMMARY: ABSTRACT.BACKGROUND: In almost every country in the world, the proportion of people aged over 60 years is growing faster than any other age group because of increased life expectancy. This demographic change has several implications for public health, especially as older age is a risk factor for many chronic diseases—diseases of long duration and generally slow progression. Chronic diseases, such as heart disease, stroke, cancer, chronic respiratory diseases, and diabetes, are by far the leading cause of death in the world, representing almost two-thirds of all deaths. Therefore in most countries, the challenge of managing increasingly ageing populations who have chronic illnesses demands an urgent response and countries such as the United States are actively researching possible solutions. ABSTRACT.WHY WAS THIS STUDY DONE?: Some studies suggest that innovations in chronic disease management that are led by nurses may help address the epidemic of chronic diseases by increasing the quality and reducing the cost of care. However, to date, reports of the evaluation of such interventions lack rigor and do not provide evidence of improved long-term health outcomes or reduced health care costs. So in this study, the researchers used the gold standard of research, a randomized controlled trial, to examine the impact of a community-based nurse care management model for older adults with chronic illnesses in the United States as part of a series of studies supported by the Centers for Medicare and Medicaid Services. ABSTRACT.WHAT DID THE RESEARCHERS DO AND FIND?: The researchers recruited eligible patients aged 65 years and over with heart failure, coronary heart disease, asthma, diabetes, hypertension, and/or hyperlipidemia who received traditional Medicare—a fee for service insurance scheme in which beneficiaries can choose to receive their care from any Medicare provider—from participating primary care practices in Pennsylvania. The researchers then categorized patients according to their risk on the basis of several factors including the number of chronic diseases each individual had before randomizing patients to receive usual care or the nurse-led intervention. The intervention included an individualized plan comprising education, symptom monitoring, medication, counseling for adherence, help identifying, arranging, and monitoring community health and social service referrals in addition to group interventions such as weight loss maintenance and exercise classes. The researchers checked whether any participating patients had died by using the online Social Security Death Master File. Then the researchers used a statistical model to calculate the risk of death in both groups. Of the 1,736 patients the researchers recruited into the trial, 873 were randomized to receive the intervention and 863 were in the control group (usual care). The researchers found that 86 (9.9%) participants in the intervention group and 111 (12.9%) participants in the control group died during the study period, representing a 25% lower relative risk of death among the intervention group. However, when the researchers considered other factors, such as sex, age group, primary diagnosis, perceived health, number of medications taken, hospital stays in the past 6 months, and tobacco use in their statistical model, this risk was slightly altered—0.73 risk of death in the intervention group. ABSTRACT.WHAT DO THESE FINDINGS MEAN?: These findings suggest that that community-based nurse care management is associated with a reduction in all-cause mortality among older adults with chronic illnesses who are beneficiaries of the fee for service Medicare scheme in the United States. These findings also support the important role of nurses in improving health outcomes in this group of patients and show the feasibility of implementing this program in collaboration with primary care practices. Future research is needed to test the adaptability, scalability, and generalizability of this model of care. ABSTRACT.ADDITIONAL INFORMATION: Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001265. This study is further discussed in a PLoS Medicine Perspective by Arlene Bierman Information about the Centers for Medicare and Medicaid Services is available The World Health Organization provides statistics on the prevalence of both chronic illness and ageing Heath Quality Partners provide information about the study BODY.INTRODUCTION: Chronically ill older adults have complex patterns of health care, frequent hospital readmissions, often receive poor or inconsistent quality of care, and account for the majority of health care expenditures in the United States [1]–[6]. Long appreciated as the dominant disease burden in upper-income countries, noncommunicable chronic disease is now recognized as a major global health problem [7],[8]. Several leading organizations and experts argue that care coordination emphasizing wellness, prevention, and chronic disease management is a promising means to increase the quality and perhaps reduce the costs of care for chronic illness in the elderly [9]–[11]. Broader testing and use of chronic disease management interventions in several countries have resulted in reports describing the challenges associated with such efforts, but very few have provided evidence of improved long-term health outcomes or reduced health care expenditures [12]–[22]. Positive reports that have been published have often come from non-experimental evaluations of clinic or practice-based interventions targeting a single chronic disease [21],[22]. Some researchers believe that innovations in nursing-led chronic disease management may help address chronic disease in areas of the world with less abundant health care resources, such as sub-Saharan Africa [23],[24]. Based on research published to date, there is uncertainty about whether nurse care management programs have the potential to improve the long-term health outcomes of chronically ill older adults. In this study we report outcomes of a longitudinal community-based nurse care management model on all-cause mortality, using a randomized controlled design. Support for this research came from the Medicare Coordinated Care Demonstration (MCCD), a national study in the United States administered by the Centers for Medicare and Medicaid Services (CMS), which sponsored 15 unique longitudinal, prospective, randomized, controlled trials [25]. Since 2002, the MCCD has independently tested these different, competitively selected, care coordination programs in an attempt to identify specific models that lower health care costs and improve quality among US Medicare beneficiaries (fee for service coverage) with chronic conditions. Descriptions of the varied programs that participated in the MCCD have been published elsewhere by Mathematica Policy Research, Inc. (MPR), the contracted evaluator for the demonstration [26],[27]. The programs selected to participate in the MCCD varied in terms of the number and types of chronic conditions they targeted. Six programs targeted a single condition, three enrolled patients using criteria other than specific diagnoses, and six targeted multiple conditions with or without additional targeting criteria. The most common primary diagnoses of patients enrolled across all programs were heart failure, coronary heart disease, and diabetes. The 15 organizations that implemented programs in the MCCD were also diverse and included four commercial disease management vendors, three hospitals, three academic medical centers, one integrated delivery system, one hospice program, one long-term care facility, one retirement community, and one health care quality research and development organization. The interventions offered by the programs in the MCCD varied, though all programs used care coordinators, which were typically registered nurses (only one program used licensed practical nurses). Nearly all programs educated patients in order to improve medication adherence, diet, exercise, and self-care. Fourteen programs sought to coordinate care for patients through a variety of mechanisms. Ten programs had timely data on hospitalizations and emergency room visits to support interventions related to the transitions of care. Fourteen programs relied on patients to provide care coordinators with a list of medications they were taking. Four programs focused on increasing physicians' adherence to evidence-based or guideline-based care. In all programs, both intervention and control participants continued to receive traditional Medicare coverage (US federal government supported fee for service payments), with an additional fixed negotiated fee per participant per month paid to the programs for each intervention participant. The impact of the various programs tested in the MCCD on medical expenditures, quality of care, and health service utilization has previously been reported [27]–[29]. The program described in the current study was designed and implemented by Health Quality Partners (HQP), (http://www.hqp.org), a not-for-profit health care quality research and development organization, and one of the programs participating in the MCCD. Some findings from the current study were previously included in a report to the US Congress, in which a 25% reduction in all-cause mortality among intervention participants compared to usual care participants was observed for the HQP program [29]. The current study was undertaken to more thoroughly evaluate the program's effect on mortality up to 5 y following enrollment. BODY.METHODS: The study protocol (Text S1) and CONSORT checklist (Text S2) are provided as supporting information. Though nested within the larger MCCD, this study's design and execution were undertaken by the authors independently of CMS or MPR. The national and HQP program-specific evaluation plans designed by MPR for the MCCD have previously been reported [30],[31]. BODY.PARTICIPANTS: All participants randomized into the HQP program from the start of the MCCD in April 2002 through March 2008 are included in this study. Traditional, fee for service Medicare beneficiaries with Parts A (hospitals, skilled nursing facility, hospice, home health care) and B (physician services, outpatient care, home health services) insurance coverage, residing in eastern Pennsylvania, 65 y of age and older, with heart failure, coronary heart disease, asthma, diabetes, hypertension, or hyperlipidemia, and receiving care at a primary care practice agreeing to work with the HQP program, were eligible to participate in this study. No minimum prior health care utilization or hospitalization was required for eligibility. Exclusion criteria included dementia, end-stage renal disease, schizophrenia, active cancer (except skin) in the prior 5 y, life expectancy less than 6 mo, and current or imminent residence in a long-term care facility. Individuals at very low risk for future health complications based on a pre-enrollment assessment were also excluded from the study. In September 2006, a protocol change made a pre-enrollment assessment of low risk an additional exclusion criterion, because interim evaluations indicated that control group participants in this stratum were not utilizing enough health care services to allow for a sufficient realization of savings in the intervention group to offset program costs. In the US, Medicare is provided in two basic forms: (1) fee for service coverage (traditional Medicare), funded and administered by the federal government, and (2) managed care coverage (Medicare Advantage), funded by the federal government, but sold and administered by private health plans. In areas of the country where insurers offer Medicare Advantage plans, Medicare beneficiaries can choose between these two types of coverage. Medicare Advantage plan members often have financial incentives to use providers within networks recognized by the health plan, and such plans may provide various forms of care coordination or chronic disease management services. By contrast, traditional Medicare beneficiaries can choose to receive their care from any participating Medicare provider and can switch providers at any time without financial penalties. Traditional Medicare, to date, lacks significant care coordination or chronic disease management benefits. All the participants in the current study were beneficiaries receiving traditional US Medicare. Potential study subjects were referred to the study from participating primary care practices. Practices were assisted to utilize administrative billing data to identify Medicare beneficiaries that might be eligible for the study based on ICD9 diagnosis codes, age, and insurance information. Primary care providers reviewed the patient list generated from billing data queries and selected patients to refer to the study. Outreach to potentially eligible patients was undertaken by HQP by way of a mailed letter of introduction and follow-up phone calls inviting referred patients to learn more about the study. BODY.DEVELOPING A NETWORK OF PARTICIPATING PRIMARY CARE PRACTICES: A network of primary care practices was developed by meeting with and describing the HQP program and the MCCD to hospitals, physician-hospital organizations, independent physicians associations, and individual practices. The basic requirements of practices agreeing to participate include: (1) responding to communications about their patients initiated by the nurse care managers on an as needed basis, (2) making the office medical records available to the nurse care managers and chart auditors, and (3) assisting in case-finding potentially eligible individuals on their patient panels, using billing system reports or extracts, or other mutually agreed to processes. The program was designed and promoted as easy to use and free of burdens related to: paperwork, recurring authorizations or pre-certifications, routine case reviews, or administrative tasks. Practices were encouraged to “test drive” the program by initially referring a small number or select set of patients meeting eligibility criteria. Offices were not required to sign a contract or commit to a minimum length of participation and there were no financial transactions involved. It was explained to offices that by virtue of the randomization process roughly half of their referred and randomized patients would be assigned to the control (usual care) group and half to the intervention group; underscoring that half of all patients from their practice that participated would not receive any extra services. Business Associate agreements committing HQP to safeguard the privacy and confidentiality of the personal health information provided by the practices were executed. During the time period of this study, 93 primary care practices in and around the 4,662-km2, four-county service area of eastern Pennsylvania (Bucks, Montgomery, Lehigh, and Northampton) agreed to participate. Patients of these practices received most of their acute care services from seven hospitals owned by six different health systems. Most practices solicited (greater than 80%) agreed to participate except for those affiliated with two hospital-owned, multi-practice networks (one operating as a Preferred Provider Organization) that declined to participate, citing their desire to: (1) implement and manage their own care coordination programs to enhance their ability to negotiate with health plans, and (2) maintain more direct control over such programs. Participating practices varied widely in terms of size (most had four or fewer primary care providers), use of electronic records, and organizational affiliation (most were independent). In the past few years, an increasing number of practices have implemented some form of the patient-centered medical home (PCMH); designed to support primary care physicians to improve the proactive coordination and tracking of patient care, typically involving the use of information systems, disease registries, and care team models. There have been no observed barriers, operational difficulties, or decreased interest in collaborating with the HQP program as the result of offices adopting the PCMH. BODY.ETHICS: CMS administered the overall conduct of the MCCD. As previously reported, “The Secretary of Health and Human Services, acting through the CMS, determined that the overall demonstration and evaluation met all criteria in both the Common Rule and National Institutes of Health's Exemption Number 5 for exemption from institutional review board review for research and demonstration projects on public benefit and service programs.” [27] (page 604). All participants provided written informed consent prior to study enrollment. HQP separately sought and received approval of the Institutional Review Board of Doylestown Hospital (Doylestown, Pennsylvania, US) for the present study. BODY.CLASSIFICATION PRIOR TO RANDOMIZATION: After providing consent, but prior to study randomization, each participant was classified using two different schema: primary diagnosis and risk stratum. The nurse care management supervisor made the determination of the primary diagnosis. For participants with only one of the chronic health conditions required for study eligibility, that condition was considered the primary diagnosis. For participants having more than one qualifying diagnosis, the condition judged most likely to precipitate a future hospitalization, on the basis of the participant's clinical measures, self-management skills, disease-specific symptoms, and hospital utilization in the prior 6 mo, was chosen as the primary diagnosis. Eligible participants were also classified into discrete categorical risk strata [31] (page 13). The first step in the algorithm HQP used to determine risk strata, is an assessment of geriatric-related risks using the Sutter Health Questionnaire (used with permission, Cheryl Phillips) [32],[33]. A number of domains are covered in this questionnaire including: self-rated health, number of medications taken, change in weight, falls, health care utilization in prior 6 mo, living arrangement, care giver status, activities of daily living, instrumental activities of daily living, ancillary health care services used, physical activity level/mobility, chronic illnesses, depression, and tobacco use. Individuals scoring at or above a level 3 on the Sutter instrument were defined as the high-risk stratum for this study. Individuals scoring below this breakpoint on the Sutter tool received a second, disease-specific risk assessment developed by HQP, which was used to classify participants into one of three additional risk strata: moderate, low, and very low. Individuals in the very low risk stratum were excluded from study participation from the outset, and those in the low-risk stratum were also excluded beginning in September 2006. In the course of administering the pre-randomization Sutter Health Questionnaire a numeric risk score (total score) was also calculated. This score was used to augment the outcomes analysis in this study by creating risk subgroups according to total score tertiles: lower, middle, and upper, defined by total scores of <15, 15–35, and >35, respectively. BODY.INTERVENTION: Participants randomized into the control group received the usual care afforded to traditional Medicare beneficiaries and following notification of their study group assignment, had no further contact with HQP. Participants randomized into the intervention group were provided the HQP model of community-based nurse care management. This model was previously described in a report by the MCCD contracted evaluators [31]. The HQP program was developed over several years in multiple care delivery settings and incorporated a broad portfolio of evidence-based preventive and care management interventions delivered longitudinally by nurse care managers in collaboration with local health care and social service providers. A detailed listing of the elements of this intervention is provided as a supplemental table (Text S3). Nurse care managers used a database developed by HQP to track their activities and participant contacts as well as key assessments and clinical data on participants. Additional paper-based documentation and assessment tools were organized and maintained in participant chart records. All intervention group participants received additional assessments to identify their physical, functional, cognitive, psychological, behavioral, social, and environmental needs. Participants determined to be in the high-risk stratum, on pre-randomization assessment, received a comprehensive, in-home geriatric assessment involving 15 specified elements, including: physical assessment (HQP), Index of Independence in Activities of Daily Living (Katz), Mini-Mental State Exam (Folstein), Clock Drawing Test (Heinik et al.), Geriatric Depression Screen-Short Form (Sheikh and Yesavage), Nutritional Risk Assessment – Nutrition Screening Initiative (NSI), violence screening (HQP), alcohol abuse screening using the CAGE Questionnaire (Ewing), behavioral and caregiver assessment, home environment safety checklist, Numeric Pain Scale (Jacox), sleep, incontinence, assessment of immunizations and preventive screenings, and psychosocial support needs (HQP). Interventions typically incorporated into an individualized plan included: education, symptom monitoring, medication reconciliation and counseling for adherence, and help identifying, arranging, and monitoring community health and social service referrals. Group interventions such as curriculum-based education; structured lifestyle and behavior change programs for weight loss; weight loss maintenance; exercise classes for improving strength and increasing physical activity; and a balance and mobility program for fall prevention were also provided directly to participants by the nurse care managers. Nurses collaborated with the participants' primary care physicians and specialists on an as needed basis to help participants achieve target clinical goals and receive appropriate and timely preventive care according to guidelines. Collaboration also allowed early identification of new or worsening conditions or symptoms, and facilitation of timely medical interventions in an effort to prevent disease exacerbation, hospital admissions, and unnecessary use of the emergency department. The nurse care managers were community based and, depending on the size of a practice's patient panel, served patients from multiple primary care practices. Participant encounters consisted of in-person visits, group sessions, and telephone contacts. In-person encounters occurred in the participants' homes, physicians' offices, and other accessible community settings, such as HQP's offices, hospitals, community centers, libraries, and faith-based organizations. Contact frequency was determined by participant need with a minimum standard of a monthly contact. On average, participants received 17.4 total contacts per year during the period included in the current study. More than half of all contacts were made in-person either as one-to-one meetings or as group classes. Individualized intervention plans were continuously updated to match the dynamic needs of participants and their caregivers. Once enrolled into the program, intervention participants received services until they died, moved out of the area, requested disenrollment, had a change in insurance coverage making them ineligible for the demonstration, or were placed in a care environment in which the nurse care manager felt they were unable to significantly add to the effectiveness of care (e.g., hospice placement). Once fully trained, each care manager served 85 to 110 participants depending on caseload complexity, geographic distribution, experience, and phase of study recruitment. In 2007, a protocol of intensified follow-up was added for participants transitioning home or to another level of care upon discharge from hospital. The protocol established guidelines by which nurses provided timely coordination and communication with hospital and post-hospital care providers. The goals were to ensure well informed, safe, and expeditious discharge plans, perform timely patient follow-through on discharge instructions, reconcile medications, and identify and address any errors, omissions, or contraindications in order to prevent readmissions and other serious adverse events. Program implementation and reliability were supported by careful nurse selection and recruitment practices, pre-service training, ongoing coaching and supervision, structured protocols, explicit operating procedures, clearly articulated performance standards, and a system of data management and statistical process control analysis and reporting to support organizational decision making. A further description of the management elements of this model is provided as a supplemental table (Text S4). This set of management practices has been described as “core implementation components” [34]. Program improvement efforts were ongoing and continuous and resulted in numerous refinements to the program over the course of its implementation within the MCCD. BODY.OBJECTIVES: The main objective of this study was to determine whether HQP's model of community-based care management, as implemented in the MCCD, is associated with a reduction in all-cause mortality overall and within subgroups of risk strata and primary diagnoses. Another objective was to determine whether there was an intervention-associated reduction in all-cause mortality within subgroups defined by tertiles of a numeric risk score obtained on intake assessment using the Sutter Health Questionnaire. The main reasons to explore treatment effect within these subgroups included: (1) refine future program eligibility criteria to direct resources to those that benefit most from the intervention, and (2) permit comparison of impacts on health outcomes to financial outcomes using similar or identical subgroups used by MPR and CMS in their separate and independent financial analyses. It was hypothesized that participants classified as belonging to one or more high-risk subgroups were more likely to demonstrate an intervention-associated reduction in mortality over the follow-up period of this study. The pre-specified and post hoc analyses of the study are summarized in Table 1. 10.1371/journal.pmed.1001265.t001Table 1BODY.OUTCOMES AND SUBGROUP ANALYSES SPECIFIED IN THE STUDY PROTOCOL.: Characteristic HQP Study Protocol Pre-specified or Post Hoc Overall Mortality (all participants) Primary outcome Pre-specified Risk stratification level Subgroup analyses Pre-specified Risk score Subgroup analyses Decision to analyze by tertile subgroups was post hoc Primary enrollment diagnosis Subgroup analyses Pre-specified Clinical cardiovascular risk factors Secondary outcomes Not part of current study because data collection is still underway BODY.OUTCOMES: PRE-SPECIFIED: The primary outcome of this study was the risk of death from any cause among intervention participants compared to control participants overall and within subgroups defined by risk strata and primary diagnosis. Vital status as of March 31, 2009 was assessed for all participants. The data source used to establish death was the online Social Security Death Master File (SSDMF) (http://www.ssdmf.com). Social security numbers obtained from participants following informed consent and prior to randomization were used to check vital status in the SSDMF. BODY.OUTCOMES: SPECIFIED POST HOC: Analyzing deaths within subgroups defined by tertiles based on the numeric risk score obtained from the Sutter Health Questionnaire was not pre-specified in the study plan. After the study began, but before analysis commenced, this outcome was added. On the basis of random samples, we estimated an overall error rate of 3%–5% in the assignment of participant risk stratum. This rate was due to mistaken Sutter level determinations resulting from the manual tallying of risk scores and variation in the optional use of “flags” (specific question responses defined in the Sutter Questionnaire), which can, if four or more are present, result in increasing the Sutter level by one level. The numeric risk score of the Sutter Health Questionnaire when calculated retrospectively by computer using questionnaire data fields was more reliable. The risk score derived from the Sutter Questionnaire is obtained in the first step of a multi-step process required for final risk stratum assignment. The risk score is a numeric variable (range in our data: 1–136, mean 29). The use of a computer calculated risk score alone, if predictive of outcomes, could offer a more streamlined, reliable, and efficient method of risk classification, potentially improving future program operations. BODY.SAMPLE SIZE: The original minimum enrollment recommendation for MCCD study sites (686 in total; 343 participants each for treatment and control groups) made by MPR as part of their sample size estimation was based on the expected impact of effective interventions on hospitalization as described in MPR's study plan for the MCCD [30]. These original sample size calculations were not based on estimated impacts on mortality. Given the actual number of overall participants in this study (1,736), the observed probability of death in the control group (0.129), and the observed unadjusted hazard ratio (0.75), with alpha set at 0.05, this study is estimated to have a power of 58% for analysis of overall mortality risk using the Cox proportional hazard method. Similarly calculated power estimates for subgroups were lower, with the exception of the upper risk tertile (power = 67%) and coronary heart disease (power = 77%) subgroups. BODY.RANDOMIZATION AND BLINDING: The study was conducted as a parallel group, randomized, controlled trial. Randomization took place at the individual participant level within each of the risk strata determined by HQP prior to enrollment (high, moderate, and low) using a secure website managed by MPR. Participants were randomized on a 1∶1 (intervention: control) basis. All randomization was done offsite by MPR per a protocol established by them and approved by CMS using randomly generated, concealed 4-digit “strings” of treatment-control assignments. By excluding strings of all treatment or all control assignments runs of more than six consecutive assignments to any group were prevented. The random assignment result was available to the program site via the website almost immediately. For practical reasons, study group assignment was not blinded. BODY.STATISTICAL METHODS: All participants randomized into the trial from its start in April 2002 through March 2008 are included in the outcome analysis according to their original study group assignment. The primary outcome (vital status) on all randomized participants (regardless of early program discontinuation) was collected and analyzed through March 31, 2009. The observation period available for each individual participant ran from his or her date of randomization through March 31, 2009 or the completion of a full 5 y of observation (whichever occurred first). Discontinuation from study participation occurring before observation endpoints were reached, for any reason including lost to follow-up, was not a reason for exclusion from the outcome analysis. Mortality over time was plotted using the Kaplan-Meier method with p-values calculated using the log-rank test. The Cox proportional hazard method was used to calculate hazard ratios. Covariates selected for inclusion in Cox regression models had a significant association with the risk of death in univariate analysis and a recognized association with mortality (sex, age group, primary diagnosis, perceived health rating, number of medications taken, hospital stays in the past 6 mo) or failed to reach significance in univariate analysis, but are widely acknowledged to have a strong association with death (tobacco use). The proportional hazard assumptions for Cox regression models were tested using Schoenfeld residuals and no violations were identified (all p-values ≥0.05). Subgroup analyses include significance testing of interaction effects using likelihood ratio testing to compare proportional hazards models with a subgroup-treatment interaction term to one without. Comparison of categorical data was performed using Fisher exact test. Comparison of continuous data was performed using the Student's t test when data was normally distributed or Wilcoxon's rank sum method when data significantly departed from a normal distribution. All values for p were calculated using two-sided tests. Statistical tests were performed using Stata/MP 10.1 for Macintosh (http://www.stata.com). BODY.RESULTS: BODY.PARTICIPANT RECRUITMENT AND FLOW: Of all patients referred (n = 9,362), sufficient data to attempt assessing study eligibility were available on 88% (n = 8,224). The CONSORT flow of participants through allocation, follow-up, and analysis is represented in Figure 1. Overall, 43% (1,736) of individuals confirmed to be eligible and living within the program service area agreed to participate in this study. Of the 2,265 (57%) eligible participants declining to participate, reasons for refusal were captured on 2,134 (94%). These are summarized in Table 2. The number of participants randomized into the study by year is presented in Table 3. 10.1371/journal.pmed.1001265.g001Figure 1BODY.CONSORT FLOW DIAGRAM.: 10.1371/journal.pmed.1001265.t002Table 2BODY.REASONS ELIGIBLE INDIVIDUALS DECLINED STUDY PARTICIPATION, :n = 2,265. Reason Number Percent Satisfied with current care 1,054 46.5 Too busy 307 13.6 Too old 230 10.2 Overwhelmed with present number of providers 195 8.6 General mistrust of solicitations 177 7.8 Initially accepted then changed their mind 171 7.5 Reason not captured 131 5.8 10.1371/journal.pmed.1001265.t003Table 3BODY.STUDY ENROLLMENT BY YEAR.: Group Year Enrolled 2002 2003 2004 2005 2006 2007 2008a Total Control 134 273 231 128 23 74 0 863 Intervention 136 276 236 128 24 73 0 873 Total 270 549 467 256 47 147 0 1,736 aThrough March 2008. Of participants randomized to the intervention group, 151 (17%) prematurely ceased receiving the intervention before reaching an observation endpoint or experiencing an outcome event (death). The most frequent reason for early withdrawal from the intervention was a change in health care insurance coverage (n = 82). Eighty-five participants in the control group also had a change in health care insurance coverage. In most cases, these changes resulted from individuals opting to enroll in a private health insurance administered Medicare plan (Medicare Advantage). The median time from enrollment to program discontinuation for any reason for intervention and control participants was 557 and 560 d, respectively. All of these participants are included in the outcome analyses. The mean follow-up for both control and intervention groups was 4.2 y. In this study, 815 (47%) participants reached the 5-y observation endpoint (alive) and 731 (42%) participants reached the March 31, 2009 endpoint (alive). Altogether, 197 (11%) died prior to reaching these endpoints. BODY.BASELINE DATA: Baseline characteristics for all participants and those belonging to the subgroups of high-risk stratum, upper risk tertile, and primary diagnosis of coronary heart disease are presented in Table 4. Overall, among the study population the mean age was 75 y, 61% were female, 31% lived alone, 17% rated their health as fair or poor, 14% said they were depressed in the prior 3 mo, and 22% reported a fall in the prior year. Participants in the high-risk stratum subgroup had an average age of 78 y, 73% were female, 67% lived alone, and 40% rated their health as fair or poor, 35% were depressed in the prior 3 mo, and 40% had fallen in the prior year. By contrast, participants in the coronary heart disease subgroup were less likely to be women (39%) and less likely to live alone (27%). Baseline characteristics of intervention and control participants are shown in Table 5 10.1371/journal.pmed.1001265.t004Table 4BODY.BASELINE CHARACTERISTICS OF PARTICIPANTS OVERALL AND SELECTED SUBGROUPS.: Characteristic Classification n (%) All n (%) High-risk Stratum Upper Risk Tertile n (%) n (%) Coronary Heart Disease Participants n 1,736 505 568 300 Sex - Female 1,057 (61) 370 (73) 416 (73) 117 (39) Age in years (mean ± SD) 74.8±6.5 78.2±7.1 78.1±6.9 76.5±6.7 Age group - years 65–69 502 (29) 85 (17) 87 (15) 61 (20) 70–74 433 (25) 78 (15) 94 (17) 66 (22) 75–79 418 (24) 130 (26) 156 (27) 72 (24) 80–84 256 (15) 124 (25) 141 (25) 72 (24) 85+ 127 (7) 88 (17) 90 (16) 29 (10) Perceived health Excellent 304 (18) 33 (7) 43 (8) 42 (14) Good 1,124 (65) 273 (54) 327 (58) 198 (66) Fair 266 (15) 161 (32) 160 (28) 56 (19) Poor 42 (2) 38 (8) 38 (7) 4 (1) Living alone 546 (31) 336 (67) 401 (71) 82 (27) Depressed in prior 3 mo 244 (14) 171 (34) 176 (31) 49 (16) Fall in prior year 374 (22) 200 (40) 210 (37) 61 (20) Limited mobility 162 (9) 153 (30) 155 (27) 34 (11) Unintended 4.54-kg+weight loss 72 (4) 41 (8) 42 (7) 11 (4) ADL score (mean ± SD) 0.8±2.1 2.2±3.4 2.0±3.3 1.0±2.5 IADL score (mean ± SD) 1.1±2.4 3.0±3.5 2.7±3.5 1.4±2.8 Need help to complete risk survey 155 (9) 119 (24) 123 (22) 31 (10) Tobacco use 80 (5) 17 (3) 18 (3) 18 (6) Chronic conditions (mean ± SD) 3.8±1.9 5.2±2.2 5.0±2.2 4.5±1.8 Nursing home stay ever in past 22 (1) 20 (4) 20 (4) 4 (1) Number of medications 5 or more 971 (56) 383 (76) 421 (74) 217 (72) 2 to 4 624 (36) 111 (22) 132 (23) 74 (25) 1 108 (6) 10 (2) 14 (2) 6 (2) None 33 (2) 1 (0) 1 (0) 3 (1) Physician or clinic visits in past 6 mo 4 or more 468 (27) 216 (43) 233 (41) 93 (31) 2 or 3 807 (46) 218 (43) 251 (44) 136 (45) 1 401 (23) 62 (12) 75 (13) 61 (20) None 60 (3) 9 (2) 9 (2) 10 (3) ER visits in past 6 mo 3 or more 19 (1) 13 (3) 14 (2) 4 (1) 2 54 (3) 39 (8) 42 (7) 15 (5) 1 240 (14) 116 (23) 126 (22) 47 (16) None 1,423 (82) 337 (67) 386 (68) 234 (78) Hospital stays in past 6 mo 4 or more 18 (1) 17 (3) 17 (3) 4 (1) 2 or 3 49 (3) 30 (6) 35 (6) 12 (4) 1 177 (10) 98 (19) 101 (18) 38 (13) None 1,492 (86) 360 (71) 415 (73) 246 (82) Primary diagnosis Heart failure 98 (6) 58 (11) 60 (11) — Coronary heart disease 300 (17) 110 (22) 116 (20) 300 (100) Diabetes mellitus 316 (18) 119 (24) 122 (21) — Hypertension 673 (39) 150 (30) 187 (33) — Asthma 81 (5) 36 (7) 39 (7) — Hyperlipidemia 268 (15) 32 (6) 44 (8) — Risk stratum High 505 (29) 505 (100) 490 (86) 110 (37) Moderate 1047 (60) — 76 (13) 170 (57) Low 184 (11) — 2 (0) 20 (7) Risk tertile Upper 568 (33) 490 (97) 568 (100) 116 (39) Middle 600 (35) 15 (3) — 111 (37) Lower 368 (33) 0 (0) — 73 (24) ADL, activities of daily living; ER, emergency room; IADL, instrumental activities of daily living; SD, standard deviation. 10.1371/journal.pmed.1001265.t005Table 5BODY.BASELINE CHARACTERISTICS OF PARTICIPANTS BY STUDY GROUP.: Characteristic Classification n (%) Intervention n (%) Control Participants 873 863 Sex - female 537 (62) 520 (60) Age in years (mean ± SD) 74.7±6.5 74.9±6.5 Age group - years 65–69 260 (30) 242 (28) 70–74 212 (24) 221 (26) 75–79 207 (24) 211 (24) 80–84 129 (15) 127 (15) 85+ 65 (7) 62 (7) Perceived health Excellent 151 (17) 153 (18) Good 566 (65) 558 (65) Fair 136 (16) 130 (15) Poor 20 (2) 22 (3) Living alone 276 (32) 270 (31) Depressed in prior 3 mo 125 (14) 119 (14) Fall in prior year 181 (21) 193 (22) Limited mobility 82 (9) 80 (9) Unintended 4.54-kg+weight loss 38 (4) 34 (4) ADL score (mean ± SD) 0.8±2.2 0.8±2.1 IADL score (mean ± SD) 1.1±2.4 1.1±2.4 Need help to complete risk survey 80 (9) 75 (9) Tobacco use 37 (4) 43 (5) Chronic conditions (mean ± SD) 3.8±1.9 3.8±2.0 Nursing home stay ever in past 12 (1) 10 (1) Number of medications 5 or more 512 (59) 459 (53) 2 to 4 301 (34) 323 (37) 1 44 (5) 64 (7) None 16 (2) 17 (2) Physician or clinic visits in past 6 mo 4 or more 232 (27) 236 (27) 2 or 3 402 (46) 405 (47) 1 206 (24) 195 (23) None 33 (4) 27 (3) ER visits in past 6 mo 3 or more 10 (1) 9 (1) 2 27 (3) 27 (3) 1 109 (12) 131 (15) None 727 (83) 696 (81) Hospital stays in past 6 mo 4 or more 7 (1) 11 (1) 2 or 3 25 (3) 24 (3) 1 90 (10) 87 (10) None 751 (86) 741 (86) Primary diagnosis Heart failure 50 (6) 48 (6) Coronary heart disease 138 (16) 162 (19) Diabetes mellitus 176 (20) 140 (16) Hypertension 348 (40) 325 (38) Asthma 39 (4) 42 (5) Hyperlipidemia 122 (14) 146 (17) Risk stratum High 252 (29) 253 (29) Moderate 528 (60) 519 (60) Low 93 (11) 91 (11) Risk tertile Upper 289 (33) 279 (32) Middle 302 (35) 298 (35) Lower 282 (32) 286 (33) ADL, activities of daily living; ER, emergency room; IADL, instrumental activities of daily living. BODY.MORTALITY ANALYSES: Overall, 86 (9.9%) intervention participants and 111 (12.9%) control participants died during the study period, representing a 25% lower relative risk of death (unadjusted hazard ratio [HR] 0.75 [95% CI 0.57–1.00], p = 0.047) among the intervention group. When covariates for sex, age group, primary diagnosis, perceived health, number of medications taken, hospital stays in the past 6 mo, and tobacco use were included in the model, the adjusted HR was 0.73 (95% CI, 0.55–0.98), p = 0.033. The number and percentages of deaths, graphical representation of the unadjusted and adjusted hazard ratios, and the p-values from tests of subgroup-treatment interaction overall, and for all subgroups are provided in Figure 2. Subgroup analyses did not demonstrate statistically significant interaction effects for any subgroup. A Kaplan-Meier plot and log rank test comparing intervention and control groups overall is shown in Figure 3. 10.1371/journal.pmed.1001265.g002Figure 2BODY.SUBGROUP ANALYSES.: Deaths and tests for interaction by subgroup. HRs and adjusted HRs (aHR) along with 95% CIs are represented by forest plots with x-axis in log 2 scale. The regression model used for the aHR includes covariates for sex, age group, primary diagnosis, perceived health rating, number of medications taken, hospital stays in the past 6 mo, and tobacco use. CHD, coronary heart disease. 10.1371/journal.pmed.1001265.g003Figure 3BODY.KAPLAN-MEIER ESTIMATE OF CUMULATIVE MORTALITY UP TO 5 Y FROM ENROLLMENT.: The plot includes results for all participants randomized into the study, (unadjusted data), with p-value calculated using the log-rank test. There was a 100% match between deaths known to nurse care managers in the intervention group and deaths identified in the Social Security Death Master File (SSDMF). BODY.ADVERSE EVENTS: No known program-related adverse events were identified. BODY.DISCUSSION: BODY.MAIN FINDINGS: This study provides evidence that the model of community-based nurse care management tested is associated with a reduction in all-cause mortality among chronically ill older adults participating in fee for service Medicare in the US. The strengths of the study include: a randomized controlled trial design (with randomization at the individual participant level), model implementation in collaboration with a broad array of primary care providers across a sizeable geographic region, a long follow-up period, and use of the intention-to-treat method of analysis. In the setting of small sample sizes and low statistical power, the subgroup analyses are best viewed as exploratory. There is a suggestion that participants in the upper risk tertile and those with a diagnosis of coronary heart disease may experience a greater survival benefit from the program. The lack of statistically significant subgroup-treatment interaction, however, indicates the need for caution when interpreting apparent differences between subgroups. The study helped confirm the feasibility of collecting self-reported information for intake risk assessment to identify subgroups that may be more likely to benefit from this intervention. The finding of an increased hazard ratio for intervention participants in the asthma subgroup was unexpected and corresponds to a total subgroup size of 81 (intervention and controls) with only three control and five intervention deaths. Retrospective reviews of HQP chart records for the five intervention participants who died revealed: one died of multiple myeloma while receiving hospice care, one of unknown causes during sleep, one of complications apparently arising from a hospital misadventure resulting in acute renal failure and sepsis, one of severe chronic obstructive lung disease while receiving hospice care, and one died more than a year after discontinuing participation in the HQP program following a change in health insurance coverage. Given the small subgroup size and event counts, Kaplan-Meier plot pattern (not shown), log rank test p-value of 0.472, and the findings noted on chart review, we believe there is little evidence of a program-related association or mechanism for an increased risk of death among intervention participants within the asthma subgroup. BODY.LIMITATIONS: CMS did not make any claims data available to HQP for program operations, performance improvement, or research purposes and did not permit HQP to have any contact with control participants following randomization. HQP's MCCD-related funding consisted of a per participant per month fee for care coordination services with no additional support for research activities associated with the demonstration. Given these limitations, the authors could not directly analyze differences in medical expenditures or health care service utilization between treatment and control groups, though these analyses have been previously reported by others [28],[29]. Evaluating the impact of care coordination models on mortality was not the primary objective of the MCCD, and the sample size for this study overall and for most subgroups was smaller than optimal for this purpose. A small sample size increases the risk of failing to identify a true difference in survival between treatment and control groups when one actually exists (a type II error), but small subgroups also increase statistical volatility such that small numbers of events or small differences in regression covariates can have an exaggerated effect on results. A likely case in point was the unexpected finding of an increased HR among intervention participants in the asthma subgroup. The study focused on one unique model of community-based care management in a single geographic region of the US. Participants in this study were predominantly white and only a small proportion was believed to be economically poor (though socioeconomic, racial, and ethnic data were not collected in this study). Testing the generalizability of this model among more racially, ethnically, culturally, and economically diverse populations and in other geographic regions is an important research imperative. Until such research is undertaken, it will be impossible to know to what extent the demographic profile of participants in the current study was a determinant of the effectiveness of the model. BODY.INTERPRETATION: The current study provides the strongest evidence to date that a model of community-based nurse care management can reduce the mortality rate for chronically ill older adults. The study also supports the broader concept that, at least under some circumstances, nurses playing a more intensive role in the longitudinal care of chronically ill older adults can improve the long-term health outcome of this population. This point had not been well established in previous research. A few studies from Europe, of smaller size and shorter duration than the study reported here, are associated with a survival advantage among older adults receiving various types of home visits by nurses [35]–[38]. To date, most studies of care coordination models in the US, including those applying the chronic care model, focusing on primary care redesign, medical homes, or transitions of care, have either not reported mortality as a separate outcome or have demonstrated no impact on mortality [39]–[53]. Two studies from the US that did not randomize individual participants and had other methodological limitations have reported improved survival for recipients of nurse care management provided within primary care settings [54],[55]. In reports by the CMS-contracted evaluators of the MCCD, the impact of the HQP model of community-based care management on health expenditures and health service utilization varied by pre-randomization risk, defined either by the risk stratification method used in the current study or by a combination of diagnoses and health service utilization. With all enrollees included (low, moderate, and high risk), no statistical difference in medical expenditures or health service utilization between the intervention and usual care groups has been observed [28],[29]. When analysis is restricted to the subgroup of the high risk stratum (as defined in the current study) intervention participants were reported to have 29% fewer hospitalizations and 20% lower expenditures than individuals assigned to usual care ([27], pages 614–615). Among a subgroup of participants with heart failure, coronary heart disease or chronic obstructive pulmonary disease, and at least one hospitalization in the prior year, the intervention group had 39% fewer hospitalizations, 37% fewer emergency room visits, a 36% decrease in total Part A and Part B Medicare expenditures and a net savings to Medicare (after HQP program fees) of US$397 per participant per month [29]. As a result of these findings, starting October 2010, with continued CMS support, eligibility for this study was changed and HQP began prospectively enrolling higher-risk beneficiaries—individuals with a history of heart failure, coronary heart disease, chronic obstructive pulmonary disease, or diabetes, and at least one hospitalization (for any reason) in the year prior to study randomization. Favorable impacts on health service utilization and expenditures among higher risk participants and reduced overall mortality suggest that this model of community-based nurse care management works by reducing avoidable complications that increase both the use of acute health care services and the risk of death. This may have been accomplished, in large part, by supporting participants to better adhere to physician-initiated treatment plans concordant with evidence-based guidelines. Nurse care managers also prompted primary care providers whenever “clinical inertia” [56] or deviations in treatment plans prevented participants from achieving guideline defined goals. Apart from being an important health outcome in its own right, improved survival, when driven, as in this case, by a preventive intervention, is very likely accompanied by other important improvements in health, functional status, and quality of life, though these have not been measured over the long-term in the MCCD. To assess the full value of this model, future research should in The HQP model of community-based care management has been tested on a regional scale in a health care delivery environment typical of much of the US for over 9 y and found to be compatible with and complementary to the work that primary care practices are increasingly engaged in to develop a patient-centered medical home. The current study also provides evidence that it is feasible to implement this program in collaboration with small, independent primary care practices. Office practices with five or fewer physicians accounted for about 73% of primary care practices in the US in 2003–2004 (with 46% of practices consisting of only one or two physicians) [57]. Smaller practices have been reported to be less likely to use patient-centered medical home processes indicating that effective chronic disease management interventions through office-based efforts alone may be especially challenging for such practices [58]. Forty-three percent of eligible individuals contacted agreed to participate in this study and 57% declined. Future experimental research must recognize and adequately accommodate the cost and time required to case-find and enroll sufficient numbers of participants into studies of this kind. Non-experimental evaluations of replication or scalability efforts, not requiring randomization or informed consent, would likely see greater rates of enrollment among those eligible. Analysis of the variation in enrollment rates between sites could potentially offer insights into how best to optimize engagement and enrollment of eligible individuals. Use of aggregated health care data, if available, would greatly improve the efficiency and effectiveness of case-finding and participant recruitment. It is not known whether the model used in the current study would be effective in other countries having different demographic profiles, socioeconomic conditions, health care insurance, or health care delivery systems. The model's attributes of being community-based, requiring relatively modest start-up capital, and its use of nurses, may make it an approach worth testing in some global health settings. The program as implemented in this study utilized collaboration with primary care physicians therefore locations with reduced availability or access to primary care services could see diminished effectiveness. In some areas, shortages of nurses with sufficient training or experience may preclude implementation of the program. Whether other types of health workers can be trained to substitute for nurses in this model is unclear, but the current study used highly experienced nurses as care managers and the use of alternative providers may not yield similar results. In light of the many care coordination and disease management models that have failed to demonstrate comparable improvements in health outcomes, it seems likely that a degree of fidelity to model design and implementation will be necessary for reproducible effectiveness. Efforts to maintain such program fidelity may conflict with the need for local adaptations to allow implementation in a new environment. The authors believe that core elements contributing to this program's effectiveness include: (1) delivering a broad set of services that match the preventive health needs of the targeted population, (2) frequent longitudinal in-person contacts with participants, (3) collaboration with primary care providers, and (4) training, management, and performance monitoring capabilities. The program of community-based care management tested in the current study appears to be a valuable addition to the primary care of appropriately selected chronically ill older adults. Efforts to more broadly test the adaptability, scalability, and generalizability of this model seem warranted. Future progress in this area will, like many innovations in biomedical science and public health, probably require multiple, well-designed, longitudinal trials. BODY.SUPPORTING INFORMATION: Text S1 Study protocol. (PDF) Click here for additional data file. Text S2 CONSORT checklist. (DOC) Click here for additional data file. Text S3 Elements of the intervention. (DOC) Click here for additional data file. Text S4 Elements of program management. (DOC) Click here for additional data file.