Ulrika Snygg-Martin
email: ulrika.snygg-martin@infect.gu.se

Rasmus Vedby Rasmussen

Christian Hassager

Niels Eske Bruun

Rune Andersson

Lars Olaison

Warfarin therapy and incidence of cerebrovascular complications in left-sided native valve endocarditis

Anticoagulant therapy has been anticipated to increase risk of cerebrovascular complications (CVC) in native valve endocarditis (NVE). This study investigates the relationship between ongoing oral anticoagulant therapy and incidence of symptomatic CVC in left-sided NVE. In a prospective cohort study CVC incidence was compared between NVE patients with and without ongoing warfarin. Among 587 NVE episodes, 48 (8%) occurred in patients on warfarin. A symptomatic CVC was seen in 144 (25%) patients with only 3 on warfarin.

CVC were significantly less frequent in patients on warfarin (6% vs. 26 %, OR 0.20, 95% CI 0.06-0.6, p=0.006).

No increase in haemorrhagic lesions was detected in patients on warfarin. Staphylococcus aureus aetiology (adjusted OR 6.3, 95% CI 3.8-10.4), and vegetation length (adjusted OR 1.04, 96% CI 1.01-1.07) were risk factors for CVC while warfarin on admission (adjusted OR 0.26, CI 95% 0.07-0.94), history of congestive heart failure (adjusted OR 0.22, 95% CI 0.1-0.52) and previous endocarditis (aOR 0.1, 95% CI 0.01-0.79) correlated with lower CVC frequency.

Introduction

Anticoagulant therapy in infective endocarditis (IE) has been controversial since the early experiences of combination therapy with antibiotics and heparin in the 1940s. A high risk of cerebral haemorrhage was seen in most studies [START_REF] Priest | The effect of anticoagulants on the penicillin therapy and the pathologic lesion of subacute bacterial endocarditis[END_REF][START_REF] Loewe | The combined use of anti-infectives and anticoagulants in the treatment of subacute bacterial endocarditis[END_REF] and the addition of anticoagulant therapy was shown unnecessary when adequate penicillin doses were administered [START_REF] Cates | Subacute bacterial endocarditis: a review of 442 patients in 14 centres appointed by the Penicillin Trials Committee of the Medical Research Council[END_REF]. Anticoagulants as a part of IE therapy was discouraged [START_REF] Finland | Current status of therapy in bacterial endocarditis[END_REF][START_REF] Pruitt | Neurologic complications of bacterial endocarditis[END_REF] but later the recommendations to some extent was modified in patients with other indications for anticoagulant therapy [START_REF] Delahaye | Cerebrovascular accidents in infective endocarditis: role of anticoagulation[END_REF].

Modern guidelines agree that IE per se is not an indication for anticoagulant therapy, but varying recommendations are given concerning ongoing anticoagulant therapy at the time of IE diagnosis [START_REF] Habib | Guidelines on the prevention, diagnosis, and treatment of infective endocarditis (new version 2009): the Task Force on the Prevention, Diagnosis, and Treatment of Infective Endocarditis of the European Society of Cardiology (ESC)[END_REF][START_REF] Baddour | Infective endocarditis: diagnosis, antimicrobial therapy, and management of complications: a statement for healthcare professionals from the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, and the Councils on Clinical Cardiology, Stroke, and Cardiovascular Surgery and Anesthesia, American Heart Association: endorsed by the Infectious Diseases Society of America[END_REF][START_REF] Westling | Swedish guidelines for diagnosis and treatment of infective endocarditis[END_REF]. Current guidelines are based on experiences from the early years of IE treatment, retrospective studies mainly involving patients with prosthetic valve endocarditis (PVE) [START_REF] Tornos | Infective endocarditis due to Staphylococcus aureus: deleterious effect of anticoagulant therapy[END_REF][START_REF] Wilson | Anticoagulant therapy and central nervous system complications in patients with prosthetic valve endocarditis[END_REF][START_REF] Karchmer | Late prosthetic valve endocarditis: clinical features influencing therapy[END_REF], and the opinions of authorities in the field [START_REF] Pruitt | Neurologic complications of bacterial endocarditis[END_REF][START_REF] Delahaye | Cerebrovascular accidents in infective endocarditis: role of anticoagulation[END_REF].

Recommendations concerning native valve endocarditis (NVE) are few [START_REF] Habib | Guidelines on the prevention, diagnosis, and treatment of infective endocarditis (new version 2009): the Task Force on the Prevention, Diagnosis, and Treatment of Infective Endocarditis of the European Society of Cardiology (ESC)[END_REF]. More recent studies addressing cerebral events in IE show that hemorrhagic complications are relatively rare in both NVE and PVE [START_REF] Ruttmann | Cerebrovascular complications in patients with left-sided infective endocarditis are common: a prospective study using magnetic resonance imaging and neurochemical brain damage markers[END_REF][14][START_REF] Thuny | Impact of cerebrovascular complications on mortality and neurologic outcome during infective endocarditis: a prospective multicentre study[END_REF] but carry a risk of excess mortality.

In our clinical experience, the risk of haemorrhagic cerebral complications in patients already on anticoagulants when IE is diagnosed is not as high as earlier described [START_REF] Delahaye | Cerebrovascular accidents in infective endocarditis: role of anticoagulation[END_REF][START_REF] Tornos | Infective endocarditis due to Staphylococcus aureus: deleterious effect of anticoagulant therapy[END_REF] and different mechanisms for embolism in native and prosthetic valve endocarditis are possible. Based on these considerations, the aim of this study was to investigate the relationship between ongoing oral anticoagulant therapy with vitamin K antagonists and the incidence of symptomatic cerebrovascular complications (CVC) in patients with left-sided NVE. A secondary aim was to evaluate clinical variables associated with CVC in NVE.

Methods

Study population

Data were obtained from 866 consecutive episodes of IE treated at one university hospital in Gothenburg (Sweden) from January 1996 to January 2008 and at two university hospitals in Copenhagen (Denmark) from October 2002 to January 2008. Left-sided NVE was seen in 587 IE episodes in 570 patients, and these episodes formed the study group. IE was classified as definite in 510 (87%) of the episodes according to the revised Duke criteria.

Patients were included prospectively in the study and demographic, microbiological, echocardiographic, radiological and clinical features were registered in a database. Patients were treated at the Cardiology Department (Denmark) or the Infectious Diseases Department (Sweden), and consultations with other specialists including thoracic surgeons, were performed regularly and, when needed, on an emergency basis.

Transoesophageal echocardiography was performed in 98% of the episodes; in the remaining episodes only transthoracic examination was possible due to early in hospital death. Presence of vegetation, maximum vegetation length and localization were registered, as well as signs of paravalvular involvement. Antibiotic and surgical therapy was given according to guidelines. Individual decisions regarding anticoagulant therapy were made by the treating physician.

Cerebrovascular complications

Patients with neurological symptoms on admission or during treatment for NVE were examined using cerebral computed tomography (CT) or magnetic resonance imaging (MRI) and lumbar puncture if indicated. Symptomatic CVC were classified as ischemic and hemorrhagic strokes, subarachnoid and intracerebral haemorrhage, transient ischemic attacks (TIA), and cerebral infections (meningitis and cerebral abscesses). . Subclinical cerebral embolism was not investigated for.

Anticoagulant therapy

Ongoing oral anticoagulant therapy was defined as prescribed, and reported continuous use of an oral anticoagulant prior to admission for IE. The only oral anticoagulant agent used among study patients was warfarin, a widely used vitamin K antagonist. To assess intensity of anticoagulation International Normalized Ratio (INR) was measured during the first day of admission in patients with ongoing warfarin therapy, but INR was not regularly measured in patients without such therapy. Discontinuation of warfarin was registered, as was replacement therapy with low molecular weight heparin.

Statistical analyses

The primary outcome variable was CVC established at admission or occurring during antibiotic treatment in patients with left-sided NVE. The patients were divided into two groups based on whether or not they were receiving warfarin at the time of admission. Between-group comparisons of categorical variables were performed using a Chi-square test or Fischer's exact test when appropriate. Continuous variables were compared using Mann-Whitney U test, and were expressed as median and interquartile (IQR) range. A multivariate logistic regression model was used to assess the independent relationship between baseline variables and CVC incidence.

Baseline variables included in the analyses were age, sex, history of diabetes mellitus, haemodialysis, congestive heart failure, previous IE or intravenous drug abuse, malignancy, immunosuppression, atrial fibrillation on admission, presence and maximum length of vegetation, affected valve (mitral versus aortic), maximum Creactive protein (CRP) level, ongoing medication with warfarin or acetylsalicylic acid (ASA) and microbiological aetiology. Variables with a univariate p-value of ≤ 0.05 were included in the multivariate analysis. Statistical analyses were performed using SPSS version 16.0.

Results

Patient characteristics

Among the 587 NVE episodes studied, 48 (8 %) occurred in patients who were receiving warfarin at the time of admission (table I). Atrial fibrillation on admission was documented in 83 out of 587 episodes of NVE (14%).

Eighteen of the 83 patients (22%) with documented atrial fibrillation on admission for NVE were on warfarin.

Warfarin use was significantly more common in episodes with atrial fibrillation at the time of NVE diagnosis than in episodes without atrial fibrillation (18/48, 38% vs. (65/539, 12%, p<0.001). Patients on warfarin had a higher prevalence of congestive heart failure prior to NVE diagnosis and tended to be older (median age 69 years vs. 64 years, p=0.058)

The prevalence of diabetes mellitus, haemodialysis and immunosuppression did not differ significantly between the groups nor did the presence or valvular localization of vegetations detected by echocardiography.

Vegetations tended to be smaller among patients on warfarin (8 vs. 10 mm, p=0.05). Frequency of cardiac surgery during antibiotic treatment did not differ significantly between the groups (46% vs. 44%) nor did inhospital mortality (10% vs. 12%). There was a male predominance in the cohort (68% men) but the proportions of men and women receiving warfarin did not differ significantly. Enterococcal NVE was significantly more frequent in the warfarin treated group (23% vs. 12%, p=0.03), but other microbiological aetiology did not differ significantly (table II).

Cerebrovascular complications

A symptomatic CVC was seen in 144 (25%) of the 587 NVE episodes, and neurological symptoms were present on admission in 74% (107/144) of these. Ischemic infarction was seen in 96 episodes (67%) and cerebral infection, mainly meningitis, in 38 episodes (26%) with CVC. Cerebral haemorrhage occurred in 14 (10%) of the episodes with a CVC, including haemorrhagic infarction in six and ruptured mycotic aneurysms in two episodes (table III). More than one type of cerebral lesion was documented in 26 out of 144 patients with CVC (18%).

Oral anticoagulants and cerebrovascular complications

CVC was seen significantly less frequently in patients on warfarin than in patients without this treatment, 6%

(3/48) vs. 26% (141/539), unadjusted odds ratio (OR) 0.2, 95% CI 0.06-0.6, p=0.006 (figure 1). The pattern with lower incidence of CVC among NVE patients on warfarin was seen for all microbiological aetiologies, but the difference was significant only for Staphylococcus aureus NVE. Of the patients with ongoing warfarin treatment suffering a symptomatic CVC, the microbiological aetiology was Staphylococcus aureus in two cases and group B streptococci in one case. Among the 48 warfarin treated patients all three CVC were already established on admission for NVE and no new CVC occurred after the start of antibiotics. Among the 539 patients not on warfarin on admission for NVE, a CVC (first time or recurrent) occurred during antibiotic treatment in 37 patients (7 %). The majority of the CVC developing after adequate antibiotic therapy was initiated were ischemic infarctions found in 29 patients, while cerebral infections were found in three patients and TIA or cerebral haemorrhage in two patients respectively.

The frequency of haemorrhagic complications among patients with and without warfarin on admission for NVE was 2 % in both groups (1/48 and 13/539, respectively). Warfarin was discontinued within the first days of IE treatment in 19 (40 %) of the episodes and replaced by low molecular weight heparin in 7 of these. One of three patients on warfarin with a CVC displayed a hemorrhagic infarction, and warfarin was discontinued on the first day of admission. Warfarin was continued in the other two patients with CVC, who were diagnosed with an ischemic lesion in one case and a brain abscess in the other. All three patients survived in-hospital treatment.

Median INR on admission was 2.0 (IQR 1.5-2.4) in the 48 patients on warfarin. INR was also registered in 80 patients not on current warfarin therapy and median INR was 1.2 (IQR 1.1-1.4) in that group.

Risk factor analysis for cerebrovascular complications

In the logistic regression analyses (table IV) ongoing warfarin therapy on admission for NVE was independently associated with a lower frequency of CVC, adjusted odds ratio (aOR) 0.26, CI 95% 0.07-0.94, p=0.04.

Staphylococcus aureus aetiology was a risk factor for CVC with an aOR of 6.3 (95% CI 3.8-10.4, p<0.001).

History of congestive heart failure was associated with fewer CVC (aOR 0.22 95% CI 0.1-0.53, p=0.001) as was a history of a previous IE (aOR 0.1, 95% CI 0.01-0.79, p=0.03). Longer vegetations detected by echocardiography correlated to a higher CVC frequency (aOR 1.04, 1.01-1.07, p=0.005) No statistical significant difference was found between maximum length of vegetation in episodes with CVC established on admission as compared with episodes with CVC occurring during antibiotic therapy (data not shown).

Discussion

Cerebrovascular events in IE are common and associated with unfavourable prognosis. Several reports on haemorrhagic side effects of anticoagulant therapy in IE patients suffering cerebral complications have been published [START_REF] Pruitt | Neurologic complications of bacterial endocarditis[END_REF][START_REF] Delahaye | Cerebrovascular accidents in infective endocarditis: role of anticoagulation[END_REF][START_REF] Tornos | Infective endocarditis due to Staphylococcus aureus: deleterious effect of anticoagulant therapy[END_REF][START_REF] Hart | Mechanisms of intracranial hemorrhage in infective endocarditis[END_REF]. Experimental studies on anticoagulant effects in IE pathogenesis are few [START_REF] Thompson | The role of bacterial adherence in the pathogenesis of infective endocarditis[END_REF][START_REF] Kroh | Von Willebrand factor-binding protein is a hysteretic conformational activator of prothrombin[END_REF] and empirical data do not support the initiation of anticoagulant therapy once IE is diagnosed. The difficult issue is dealing with ongoing anticoagulation therapy at the time of diagnosis of IE. Recommendations concerning this are based on low level evidence in guidelines dealing with the management of IE [START_REF] Habib | Guidelines on the prevention, diagnosis, and treatment of infective endocarditis (new version 2009): the Task Force on the Prevention, Diagnosis, and Treatment of Infective Endocarditis of the European Society of Cardiology (ESC)[END_REF][START_REF] Westling | Swedish guidelines for diagnosis and treatment of infective endocarditis[END_REF] Therefore, clinicians have little information available to inform a decision regarding continuation of anticoagulation in this situation.

However, the number of individuals on anticoagulant therapy is rising [START_REF] Go | Prevalence of diagnosed atrial fibrillation in adults: national implications for rhythm management and stroke prevention: the AnTicoagulation and Risk Factors in Atrial Fibrillation (ATRIA) Study[END_REF]. The present study addresses the relationship between ongoing oral anticoagulant therapy during development of NVE and the incidence of CVC in a large prospective cohort. This design was intended to eliminate confounding from inherent differences in CVC rate between NVE and PVE. Since IE is a relatively rare disease a randomized trial to evaluate this question is not possible. The majority of cerebral complications have already occurred when IE is diagnosed (74% in the present series) and the intention was not to evaluate the effect of initiation of warfarin therapy after NVE diagnosis, which would be better addressed in a randomized trial.

After multivariate modelling a lower risk of CVC prevailed in patients with ongoing warfarin treatment on admission for NVE (aOR 0.26, 95% CI 0.07-0.94, p=0.04) compared to patients not on warfarin. The difference was seen among non-hemorrhagic intracerebral events, while hemorrhagic events were uncommon in both groups. A lower rate of CVC in anticoagulated patients with NVE contradicts the results in a previous retrospective study separately addressing the issue of cerebrovascular accidents in NVE patients on anticoagulant therapy [START_REF] Delahaye | Cerebrovascular accidents in infective endocarditis: role of anticoagulation[END_REF]. In that study cerebral bleeding was seen in 5% of the NVE patients and infarctions in 7%, which is a relatively low number of ischemic events compared to other series. A decreasing risk of intracranial haemorrhage over time in PVE patients on anticoagulant therapy has been found [START_REF] Leport | Fifty cases of late prosthetic valve endocarditis: improvement in prognosis over a 15 year period[END_REF], contrary to the increasing frequency of anticoagulant associated intracerebral haemorrhage reported during the 1990s in patients with anticoagulant therapy for any indication [START_REF] Flaherty | The increasing incidence of anticoagulant-associated intracerebral hemorrhage[END_REF].

The frequency of cerebral haemorrhage was low among our patients, which is in accordance with recent IE studies [START_REF] Thuny | Impact of cerebrovascular complications on mortality and neurologic outcome during infective endocarditis: a prospective multicentre study[END_REF][START_REF] Heiro | Neurologic manifestations of infective endocarditis: a 17-year experience in a teaching hospital in Finland[END_REF]. The high proportion of anticoagulant associated cerebral haemorrhages found by Tornos et al. [START_REF] Tornos | Infective endocarditis due to Staphylococcus aureus: deleterious effect of anticoagulant therapy[END_REF] in Staphylococcus aureus PVE was not reproduced in this study of NVE. The median INR the first day of admission was 2.0 so almost half of the values were lower than the therapeutic range (2.0-3.0). This might have contributed to some degree to the low frequency of cerebral hemorrhagic complications seen among our patients on anticoagulant therapy.

After admission for IE, warfarin was continued in 60% of the patients, discontinued in 40%, and replaced with low molecular weight heparin in one third of these. Irrespective of what decision was taken regarding the warfarin treatment no CVC was seen in the warfarin group after the initiation of antibiotic treatment. Among patients not on warfarin, the CVC frequency after initiation of antibiotic treatment was 7%. The low frequency of cerebral emboli after initiation of antibiotics is well known [START_REF] Dickerman | The relationship between the initiation of antimicrobial therapy and the incidence of stroke in infective endocarditis: an analysis from the ICE Prospective Cohort Study (ICE-PCS)[END_REF] and emphasizes that early diagnosis of IE is of major importance to reduce the number of cerebral complications.

The definition of CVC used in this study was based on the assumption that both infectious and non-infectious cerebral complications in IE have a common pathogenetic pathway through septic embolism from vegetations.

Ischemic stroke with focal neurological symptoms is the most frequently reported cerebral complication in IE but also well-documented are more complex syndromes with varying combinations of focal neurological signs, meningism and encephalopathy in the same patient as well as silent cerebral embolism [START_REF] Pruitt | Neurologic complications of bacterial endocarditis[END_REF]14,[START_REF] Cam | Neurologic complications in a group of 86 bacterial endocarditis[END_REF][START_REF] Cooper | Subclinical brain embolization in left-sided infective endocarditis: results from the evaluation by MRI of the brains of patients with left-sided intracardiac solid masses (EMBOLISM) pilot study[END_REF]. Autopsy data have indicated that embolic meningoencephalitis is the fundamental neuropathological change in IE [26] and a continuum between vascular and infectious cerebral complications in endocarditis has been suggested [27,[START_REF] Bitsch | Focal neurologic deficits in infective endocarditis and other septic diseases[END_REF] Staphylococcus aureus aetiology was a major risk factor for CVC in our study as has been shown previously [START_REF] Heiro | Neurologic manifestations of infective endocarditis: a 17-year experience in a teaching hospital in Finland[END_REF]. Vegetation length was associated with a higher frequency of cerebral complications, but since most CVC were established before the first echocardiographic examination was performed, the implications of this finding to reduce the number of CVC in IE patients are limited. However, vegetation length can also be used as a prognostic marker in IE and has in other studies been correlated to an increased frequency of embolic events during antibiotic treatment [START_REF] Thuny | Risk of embolism and death in infective endocarditis: prognostic value of echocardiography: a prospective multicenter study[END_REF]. Median vegetation length in patients on warfarin was 8 mm as compared with 10 mm in patients not using warfarin (p=0.05). Warfarin induced lower levels of vitamin K dependent coagulation factors might impact on the formation and dynamics of vegetations and give a protective effect on CVC risk through this mechanism. Lower rates of embolism has also been observed in PVE patients [START_REF] Mangoni | Risk factors for "major" embolic events in hospitalized patients with infective endocarditis[END_REF], where most patients are on warfarin at the time of IE. Additional beneficial influence of warfarin treatment on cerebral embolic risk can be hypothesized, e.g. through separate fibrin interactions [START_REF] Thompson | The role of bacterial adherence in the pathogenesis of infective endocarditis[END_REF]. Another indirect effect of warfarin treatment is regular INR follow-up, which might shorten the duration of symptoms before admission for NVE. A history of congestive heart failure and previous IE episode was associated with a lower risk of CVC in the study cohort. Possible explanations for this could be better access to health care facilities and higher diagnostic awareness of concomitant cardiac conditions in this type of patients. However, the observed relation between history of congestive heart failure and lower CVC incidence among NVE patients is of unknown significance and could represent confounding due to variables not registered (i.e. use of other cardiac medication such as statins or angiotensin-converting enzyme inhibitors) or a chance finding. Regarding individuals who have experienced a previous IE shorter diagnostic delay is possible.

Patients on warfarin tended to be older and had a higher frequency of enterococcal NVE as compared with patients not on warfarin. Fewer CVC among warfarin treated patients was seen for all microbiological aetiologies, but the difference was significant for episodes caused by Staphylococcus aureus only, as separately described [START_REF] Rasmussen | Major cerebral events in Staphylococcus aureus infective endocarditis: is anticoagulant therapy safe?[END_REF]. This uniform pattern irrespective of NVE causative organism supports the results of the multivariate analysis with an independent association between warfarin use and lower CVC incidence. Warfarin treated patients had also a higher frequency of atrial fibrillation on admission. This was expected since atrial fibrillation is an important indication for anticoagulation. Among patients with atrial fibrillation, only one fifth were on warfarin on admission for IE. This figure might reflect a high proportion of new atrial fibrillation induced by fever or heart failure associated with the current NVE episode or patient characteristics not elucidated in the study. Atrial fibrillation was not significantly associated with cerebral emboli among our patients, but the power of the present study was insufficient to evaluate differences in an expected excess embolic frequency of 5-10% per year.

Several limitations of our study must be acknowledged. First, this is a non-randomized observational study and despite prospective enrolment unmeasured variables may bias our results. Second, since the number of patients on warfarin treatment is relatively low and the number of events in this group is only three, misclassification or other bias might influence the results of the analyses. Therefore, our findings must be considered preliminary and revaluated in other studies. Third, individual indications for warfarin was not registered in the study protocol and during the 12-year study period the use of anticoagulation in patients with NVE may have changed. Fourth, the generalisability to other NVE populations must be questioned, since warfarin treatment and follow-up as well as IE treatment and prognosis is influenced by multiple factors, such as health care accessibility, age structure and health status of the population at risk, microbiological resistance patterns and proportion of surgically treated patients. Previous studies have shown relatively low IE related in-hospital mortality rates in Scandinavian populations [START_REF] Olaison | Early surgery in infective endocarditis[END_REF] as compared with mortality in other regions of the world [START_REF] Murdoch | Clinical presentation, etiology, and outcome of infective endocarditis in the 21st century: the International Collaboration on Endocarditis-Prospective Cohort Study[END_REF]. Influence of referral bias also limits the comparability of results in different studies.

In conclusion, there was no increased risk of haemorrhagic complications in patients with NVE continued on warfarin in this study population. This finding needs to be confirmed by other investigators before more specific recommendations can be made but suggests that clinicians may cautiously continue ongoing anticoagulation in patients with NVE with or without CVC other than haemorrhagic brain lesions. The reduced risk of ischemic cerebral complications with ongoing anticoagulation in patients with left-sided NVE also needs to be confirmed by other investigators.

34.

Figure I .

 I Figure I. Frequency of cerebrovascular complications in 587 left-sided native valve endocarditis episodes in patients with or without warfarin.

Table I .

 I Clinical characteristics among patients with native valve endocarditis. Groups divided by use of warfarin on admission.

		Not using warfarin	Using warfarin
	Variable	N=539 (%)	N=48 (%)	P
	Female sex	175 (32)	15 (31)	> .30
	Definite infectious endocarditis	469 (87)	41 (85)	> .30
	Age (median) years (IQR)	64 (50-75)	69 (59-77)	0.058
	Diabetes mellitus	66 (12)	7 (15)	> .30
	Haemodialysis	23 (4)	2 (4)	> .30
	History of congestive heart failure	77 (14)	15 (31)	0.002
	Previous infective endocarditis	29 (5)	4 (8)	> .30
	Atrial fibrillation on admission	65 (12)	18 (38)	<.001
	History of malignancy	42 (8)	2 (4)	> .30
	Immunosuppression	56 (10)	8 (17)	0.18
	Intravenous drug use	23 (4)	1 (2)	> .30
	Mitral valve involvement	289 (54)	27 (57)	> .30
	Aortic valve involvement	297 (55)	26 (54)	> .30
	Vegetation detected by echocardiography	409 (79)	37 (78)	> .30
	(N=562)
	Length of vegetation (median) mm (IQR)	10 (5-14)	8 (4-11)	0.05
	CRP maximum level (median) mg/L (IQR)	118 (68-206)	119 (60-180)	> .30
	Receiving acetylsalicylic acid	122 (23)	7 (15)	0.20
	Surgery	238 (44)	22 (46)	> .30
	Mortality	68 (12)	5 (10)	> .30
	CRP: C-reactive protein, IQR: interquartile range

Table II .

 II Microbiological aetiology in 587 left-sided native valve endocarditis episodes in patients with or without warfarin on admission.

		Not using warfarin N=539	Using warfarin N=48 (%)
	Variable	(%)		P
	Staphylococcus aureus	132 (24)	10 (21)	> .30
	Viridans group streptococci	183 (34)	17 (35)	> .30
	Enterococci	65 (12)	11 (23)	0.03
	Other streptococci	39 (7)	3 (6)	> .30
	Coagulase-negative staphylococci	32 (6)	2 (4)	> .30
	Blood culture negative	57 (11)	4 (8)	> .30
	Miscellaneous	31 (6)	1 (2)	0.28

Table IV .

 IV Variables associated with cerebrovascular complications in 587 episodes of native valve endocarditis.Univariate and multivariate risk factor analysis OR: odds ratio, CI: confidence interval, NVE: native valve endocarditis, IE: infective endocarditis, CRP: C-

		Univariate logistic regression	Multivariate logistic regression
		OR (95 % CI)	P	Adjusted OR (95 % CI)	P
	Variable
	Staphylococcus aureus	5.8 (3.8 -8.8)	<0.001	6.3 (3.8 -10.4)	<0.001
	Receiving warfarin at NVE	0.2 (0.06 -0.6)	0.006	0.26 (0.07 -0.94)	0.04
	diagnosis
	History of congestive heart	0.3 (0.14-0.59)	0.001	0.22 (0.1 -0.52)	0.001
	failure
	Previous IE episode	0.1 (0.01 -0.66)	0.018	0.1 (0.01 -0.79)	0.03
	Vegetation detected by	2.5 (1.5 -4.4)	0.001	-	-
	echocardiography
	Length of vegetation	1.05 (1.03 -1.08)	<0.001	1.04 (1.01 -1.07)	0.005
	(median)
	CRP maximum level	1.004 (1.00 -1.006)	<0.001	1.0 (0.99 -1.01)	0.20
	(median)
	Female sex	1.3 (0.9-1.9)	0.19	0.9 (0.54 -1.4)	>0.30
	Age	0.99 (0.98-1.01)	>0.30	0.99 (0.98-1.01)	>0.30
	reactive protein

Toone CE (1941) Cerebral manifestations of bacterial endocarditis. Ann Intern Med 14:1551-1574

Acknowledgement

We thank Salmir Nasic of the Research and Development Centre, Skaraborg Hospital, for assistance with the statistical analyses.


Dr Simone Guglielmetti
email: simone.guglielmetti@unimi.it.

Prof. Dr Diego Mora

Martin Gschwender

Katja Popp

Sandra Hug

Dr. med Martin Gschwender

significantly alleviates irritable bowel syndrome and improves quality 2 of life: a double-blind, placebo-controlled study 3 4

Keywords: Irritable bowel syndrome < Disease-based, Probiotics/prebiotics < Topics, X keyword = no topic, Y keyword Irritable Bowel Syndrome, IBS, Colon irritable, Intestinal microbiota, 48 Probiotics, Bifidobacteria 49 50 51 52 53 54 55

come

Irritable bowel syndrome (IBS) is a common gastrointestinal functional disorder, 57 diagnosed through the Rome III criteria [START_REF] Longstreth | Functional bowel disorders[END_REF]. Abdominal pain, flatulence and bloating 58 are common IBS symptoms for which no endoscopic, biochemical or radiological 59 cause is verifiable. IBS affects 15-20 % of the population, with a highly increasing 60 tendency in industrial nations [START_REF] Madden | A review of the role of the gut microflora in irritable 399 bowel syndrome and the effects of probiotics[END_REF][START_REF] Silk | Clinical trial: the effects of a trans-galactosaccharide prebiotic on 403 faecal microbiota and symptoms in irritable bowel syndrome[END_REF][START_REF] Brenner | The Utility of probiotics in the treatmet of irritable bowel 407 syndrome: a systematic review[END_REF]. The pathophysiology of IBS is yet only partly 61 understood. Recent research suggests that an imbalance of the intestinal microbiota 62 with a significant reduction of bifidobacteria and a dysfunctional intestinal barrier with 63 subsequent bacterial translocation may contribute to the development of IBS and its 64 symptoms [START_REF] Pimental | Eradication of small intestinal bacterial overgrowth reduces 411 symptoms of irritable bowel syndrome[END_REF][START_REF] Zeng | Clinical trial: effect of active lactic acid bacteria on mucosal barrier 415 function in patients with diarrhea-predominant irritable bowl syndrome[END_REF][START_REF] Marshall | Intestinal permeability in patients with irritable bowel syndrome 419 after a waterborne outbreak of acute gastroenteritis in Walkertin, Ontario[END_REF][START_REF] Kassinen | The fecal microbiota of irritable bowel syndrome patients differs 423 significantly from that of healthy subjects[END_REF][START_REF] Mättö | Composition and temporal stability of gastrointestinal microbiota in 426 irritable bowel syndrome -a longitudinal study in IBS and control subjects[END_REF]. 65

As probiotics have been reported in several studies to restore the intestinal 66 microbiota and gut barrier as well as hinder bacterial translocation, the therapeutic 67 potential of probiotics in IBS has become of strong interest [START_REF] Alvares-Olmos | Probiotic agents and infectious diseases: a 430 modern perspective on a traditional therapy[END_REF][START_REF] Salminen | Clinical use of probiotics for stabilizing the gut mucosal barrier: 434 successful strains and future challenges[END_REF]. However the 68 efficacy of probiotics is strongly strain specific and only certain strains might be able 69 to improve IBS and its symptoms [START_REF] Fanigliulo | Role of gut microflora and probiotic effects in the irritable bowel 438 syndrome[END_REF][START_REF] O'mahony | Lactobacillus and Bifidobacterium in irritable bowel syndrome: 441 symptom responses and relationship to cytokine profiles[END_REF][START_REF] Kajander | A probiotic mixture alleviates symptoms in irritable bowel 445 syndrome patients: a controlled 6-month intervention[END_REF]. The ability of specific strains to adhere 70 well to intestinal cells may play a pivotal role in altering the intestinal microbiota and 71 increasing the intestinal barrier, which might be of significant value especially in the 72 treatment of IBS. Guglielmetti et al. were able to show in a Caco-2 cell line that the 73 adherence of B. bifidum MIMBb75, which was isolated from a fecal sample of a 74 healthy adult, was significantly better than that of well-studied commercial probiotics 75 [START_REF] Guglielmetti | Implication of an outer surface lipoprotein in adhesion of 449 bifidobacterium bifidum to caco-2 cells[END_REF][START_REF] Guglielmetti | Study of the adhesion of bifidobacterium bifidum MIMBb75 453 to human intestinal cell lines[END_REF]. Taking into consideration the pathophysiology of IBS and the role of 76 adherence of probiotics regarding the restoration of the intestinal microbiota as well 77 as of the gut barrier, B. bifidum MIMBb75 might be effective for IBS treatment. As the 78 conventional medical treatment of IBS is unsatisfactory, we have evaluated the 79 efficacy of B. bifidum MIMBb75 in IBS in a double blind, placebo-controlled multi-80 center study. 81 presented to the Ethics Committee of the Bavarian Chamber of Physicians. For 88 inclusion, subjects aged between 18 and 68 years with mild to moderate IBS (Rome 89 III criteria) have been considered. Individuals with inflammatory organic 90 gastrointestinal disease, systemic diseases, cancer, autoimmune diseases, diabetes, 91 known lactose intolerance or immunodeficiency, known further abdominal surgery 92 except appendectomy, being older than 50 years and having had a positive 93 sigmoidoscopy or coloscopy within the last five years, diagnosed hyperthyroidism, 94 use of antipsychotics or systemic corticosteroids for at least 3 months prior to study 95 start, major psychiatric disorder, celiac disease or pregnancy had been excluded. 96 97

Study Design 98

This study was performed as a prospective, multi-center, randomized, double-blind, 99 placebo-controlled, two-arm nutritional study. 100 Throughout the study, patients recorded their global IBS symptoms on a daily basis 101 as well as individual IBS symptoms using a patient diary. Additionally, patients have 102 been questioned at a physician site for IBS symptoms (visit 2-4) and quality of life 103 (visit 3 and 4). 104

Physician visits took place at screening, after two weeks (run-in phase), after 6 105 weeks (end of treatment) and after 8 weeks (end of wash-out phase) (Figure 1). 106 After the nutritional supplement-free wash-out phase (visit 4, day 57), a complete 126 physical examination was performed and a blood sample was taken. "distension/bloating", and "urgency", recorded on the same 7-point Likert scale. The 163 individual symptom scores were additionally combined into a composite symptom 164 score as the arithmetic mean of three individual symptom scores. Furthermore, the 165 number of bowel movements, feeling of incomplete bowel evacuation and intake of 166 other medications were reported daily in the diary. 167

At the end of the treatment and again at the end of the study, physicians questioned 168 the patients regarding the global assessment of efficacy, tolerability as well as 169 digestive disorder ("bowel movement satisfaction"). Efficacy was assessed by the 170 following question: "Please consider how you felt during the 4 week treatment 171 regarding your overall well-being, and symptoms of abdominal discomfort/pain and 172 altered bowel habit. Compared to the way you usually felt before taking the study 173 medication, how would you rate your relief of symptoms during the last 4 weeks?" 174 Possible answers were: "completely relieved (1), considerably relieved (2), somewhat 175 relieved (3), unchanged (4) or worse (5)". Both "completely relieved" and 176 "considerably relieved" were defined as "adequate relief". 177 Health related quality of life was assessed by the use of the SF-12 questionnaire 178 prior to the treatment and at the end of the treatment. 179

Adverse events were recorded throughout the study and the global assessment of 180 tolerability was questioned at physician visit 3 and 4. Laboratory values and vital 181 signs were examined at the screening visit and at the end of the study. 182

Statistical analysis 197

The primary objective of this study was to prove a significant reduction of the SGA of 198 general IBS symptoms at the end of the treatment in the bifidobacteria group vs. 199 placebo. The SGA was calculated for each subject as arithmetic mean at baseline, 200

during the treatment period and during the wash-out phase. To account for possible 201 differences in the baseline values, the change from baseline calculated as mean 202 score during 4 weeks of treatment minus mean score during the run-in phase (week 203 1-2) was defined as primary target criterion. The non-parametric Van Elteren test 204 stratified by study centers was used for the comparison of treatment arms. P<0.05 205 was considered statistically significant. 206

The primary analysis was based on the intent-to-treat population where all 207 successfully randomized patients were included. Missing post-baseline values were 208 imputed by the baseline value for the primary target criterion and these patients were 209 evaluated as non-responders (n=1 during treatment, n=3 during wash-out phase). An 210 additional per protocol analysis was performed for supportive purposes. 211 In terms of baseline characteristics, there were no significant differences between the 234 groups. 21.3% were classified as diarrhea-predominant IBS (23.3% 2.07 in the treatment phase) compared to only -0.16 points [95%CI: -0.32; 0.00] (from 249 2.79 in the run in phase to 2.63 in the treatment phase) in the placebo group 250 (p<0.0001) using the 7-point Likert scale. The evaluation of the SGA on a weekly 251 basis showed a significant benefit for patients within the bifidobacteria group for 252 every single week starting the second week of treatment till the end of the study (Fig. 253 3). 254 255

Secondary endpoints 256

Secondary endpoints included changes in IBS symptoms -"pain/discomfort", 257 "distension/ bloating", "urgency", "number of bowel movements" and "feeling of 258 (p=0.0001) during the treatment but not during the wash-out phase. No effects could 266 be detected for frequency of bowel movement and feeling of incomplete bowel 267 evacuation (Fig. 4). 268

269

The evaluation of the IBS symptoms pain/discomfort and distension/bloating on a 270 weekly basis showed a significant benefit for patients within the bifidobacteria group 271 compared to placebo for every single week beginning the second week of treatment 272 till the end of the study. A significant difference in urgency between the bifidobacteria 273 and the placebo group was shown between week four and six (Fig. 5 and Fig. 6). 274 275 Bowel movement satisfaction (digestive disorder) decreased from 3.89 to 2.44 in the 276 bifidobacteria group vs. 3.69 to 3.47 in the placebo group (p=0.0002) after treatment. 277

The reduction persisted during wash-out phase (2.33 Overall responders were defined as patients experiencing an improvement of the 293 average weekly score of at least 1 point on the Likert scale for the primary parameter 294 (SGA of IBS symptoms) in at least two out of the 4 weeks treatment period (50% 295 rule). Abdominal pain responders were defined using the same 50% rule for at least 296 one point average improvement for the assessment of "pain/discomfort". Overall 297 responder rates were 56.7% in the bifidobacteria group and only 21.0% in the 298 placebo group (p=0.0001). The difference between the treatment arms was only a 299 little bit less pronounced when considering only the symptom "pain/discomfort" where 300 responder rates were calculated to be 48.3% in the bifidobacteria and only 24.2% in 301 the placebo group (p=0.008) (Fig. 7). 302 303

Global efficacy at physician site 304

The overall assessment of efficacy was significantly better in the bifidobacteria group 305 compared to placebo. At the end of treatment 43.3% of the patients in the 306 bifidobacteria group achieved adequate relief compared to only 8.1% in the placebo 307 group (p<0.0001). At the end of the study adequate relief was reported for 46.7% in 308 the bifidobacteria and 11.3% of the patients in the placebo group (p<0.0001; Fig. 8). 309 310

Health related quality of life 311

The evaluation of the SF12 sum scores showed a significant gain in quality of life 312 within the bifidobacteria group. Physical health sum improved from 47. 89 Up to date, several studies have examined the effects of probiotics on IBS and its 343 symptoms [START_REF] O'mahony | Lactobacillus and Bifidobacterium in irritable bowel syndrome: 441 symptom responses and relationship to cytokine profiles[END_REF][START_REF] Kajander | A probiotic mixture alleviates symptoms in irritable bowel 445 syndrome patients: a controlled 6-month intervention[END_REF][START_REF] Williams | Clinical trial: a multistrain probiotic preparation significantly 460 reduces symptoms of irritable bowel syndrome in a double-blind placebo-controlled 461 study[END_REF][START_REF] Guyonnet | Effect of fermented milk containing Bifidobacterium animalis 464 DN-173 010 on the health-related quality of life and symptoms in irritable bowel 465 syndrome in adults in primary care: a multicentre, randomized, double-blind, 466 controlled trial[END_REF]. However, only a few could show a significant benefit. 344 Additionally, to our knowledge, no probiotic strain could show to significantly alleviate 345 irritable bowel syndrome and simultaneously improve the quality of life. While some 346 studies might have missed to show efficacy due to small sample size and 347 randomization errors, several different probiotic strains did repeatedly show no 348 significant improvement in IBS [START_REF] Marteau | Probiotics in functional intestinal disorders and IBS: proof of action 469 and dissecting the multiple mechanisms[END_REF][START_REF] Bausserman | The use of Lactobacillus GG in irritable bowel 472 syndrome in children: a double-blind randomized control trial[END_REF][START_REF] Niv | The efficacy of Lactobacillus reuteri ATCC 476 55730 in the treatment of patients with irritable bowel syndrome-a double blind, 477 placebo-controlled, randomized study[END_REF][START_REF] Kim | A randomized controlled trial of a probiotic combination VSL#3 and 480 placebo in irritable bowel syndrome with bloating[END_REF][START_REF] Sen | Effect of Lactobacillus plantarum 299v on colonic fermentation and 484 symptoms of irritable bowel syndrome[END_REF]. Recently, Brenner et al. ( 2009) 349 published a systematic review of randomized controlled trials (RCTs) aimed at the 350 evaluation of the efficacy, safety, and tolerability of probiotics in the treatment of IBS. 351 A total of 16 RCTs were included in the analysis. Of those, exclusively one 352 bifidobacteria strain showed efficacy for improvement of IBS symptoms in two 353 appropriately designed studies [START_REF] Brenner | The Utility of probiotics in the treatmet of irritable bowel 407 syndrome: a systematic review[END_REF]. Apart from inappropriate study design, in our 354 opinion, these findings could be attributed to the fact that the efficacy of probiotics is 355 strongly strain specific and that only few strains might be able to show efficacy in 356

IBS. 357

No study to date could prove unambiguously the mode of action of probiotics, which 358 can be clearly linked to the improvement of IBS and its symptoms. Several studies 359 have observed altered intestinal microbiota with a significant lack of bifidobacteria 360 [START_REF] Kerckhoffs | Lower Bifidobacteria counts in both duodenal mucosa-488 associated and fecal microbiota in irritable bowel syndrome patients[END_REF][START_REF] Si | Intestinal microecology and quality of life in 492 irritable bowel syndrome patients[END_REF] and a dysfunctional intestinal barrier in IBS patients. These studies have 361 been supported by the fact that a normalization of the lactulose breath test, which 362 suggests the presence of small intestinal bacterial overgrowth or an increased 363 number of enteric microorganisms, is highly correlated with a significant reduction of 364 IBS symptoms [START_REF] Pimental | Normalization of lactulose breath testing correlates with 496 symptom improvement in irritable bowel syndrome: a double-blind, randomized, 497 placebo-controlled study[END_REF]. It has been stated that the imbalance of the microbiotic 365 composition may lead to a different fermentation pattern, especially with increased 366 hydrogen production resulting in bloating [START_REF] Keith | Do interventions which reduce colonic bacterial 501 fermentation improve symptoms of irritable bowel syndrome?[END_REF][START_REF] Quigley | Probiotics in irritable bowel syndrome: an immunomodulatory 505 strategy[END_REF]. Additionally, gut mucosal barrier 367 disruption has been proven to be associated with bacterial translocation and 368 inflammatory conditions [START_REF] Brenner | The Utility of probiotics in the treatmet of irritable bowel 407 syndrome: a systematic review[END_REF][START_REF] Pimental | Eradication of small intestinal bacterial overgrowth reduces 411 symptoms of irritable bowel syndrome[END_REF][START_REF] Zeng | Clinical trial: effect of active lactic acid bacteria on mucosal barrier 415 function in patients with diarrhea-predominant irritable bowl syndrome[END_REF]. It seems likely that only specific strains are able to 369 restore an imbalanced intestinal microbiota and gut barrier, however further research 370 is needed to investigate if the positive effects of B. bifidum MIMBb75 can be 371 attributed to this mode of action. 372

There are several limitations of this study. First of all, the study population was not 373 large enough for a sub-group analysis of IBS subtypes. We could therefore not show 374 whether some IBS-subtypes would benefit more from the consumption of B. bifidum 375 MIMBb75 than others. In order to prove whether some subgroups might benefit 376 more, a larger study might be useful. The study population has mild to moderate IBS 377 symptoms as evidenced by the indicated SGA-baseline values. A further study 378 should be aimed at the examination whether the patients with severe IBS symptoms 379 will profit in the same way as those with mild to moderate symptoms or whether these 380 patients are more refractory to this treatment. 381

Additionally, the duration of the study was perhaps short at 4 weeks. It would be of 382 specific value to explore whether patients benefit more from a longer consumption of 383 B. bifidum MIMBb75. Finally, the observation that patients still benefit during the 384 consumption-free wash-out phase is of significant value and requires further 385 investigation (for longer follow-up). 386

In conclusion, the beneficial effects of B. bifidum MIMBb75 in improving global IBS as 387 well as its symptoms along with the good side effect profile suggests that this 388 probiotic strain has the potential as an effective alternative to current treatment 389

  in several physician centers in Bavaria, Germany from 86 principal investigators and by advertisements. The nutritional study protocol has been 87

  have given their written informed consent, they qualified for the 108 screening examination on day 1 (visit 1), which included a complete medical history 109 and physical examination. A blood sample was taken for analysis in a central 110 laboratory, including a pregnancy test. At the screening visit patients were instructed 111 to maintain their eating and life style habits throughout the study. A patient diary was 112 handed out. 113At the second visit (day 15) diaries were reviewed. Patients who had at least 2 days 114 with mild to moderate pain during the second week of run-in and who fulfilled all 115 inclusion criteria and who did not violate any of the exclusion criteria were 1:1 116 randomized to receive either B. bifidum MIMBb75 or placebo. The treatment was 117 allocated according to a computer-generated blocked randomization list with a block 118 size of 4. The block size was not disclosed to the investigators. During the 119 intervention period, patients received either one probiotic capsule daily over a 4 week 120 period or an identical appearing placebo. The allocation was blinded to both patients 121 and site staff. 122At the end of the treatment phase (visit 3, day 43), investigators collected the unused 123 study product and empty sachets in order to confirm compliance. Diaries were 124 collected and reviewed. 125

8 A

 8 reduction of the subject's global assessment (SGA) of at least 20% on the 7-point 186 Likert scale was considered as a relevant treatment effect. Based on published data 187 (17), a difference of 0.6 points in the SGA of IBS symptoms between B. bifidum 188 MIMBb75 and placebo on the 7-point Likert scale was anticipated (e.g. 3.0 in the 189 placebo group and 2.4 in the bifidobacteria group). Standard deviation was estimated 190 with 1.0 using the same data. With these assumptions, a Wilcoxon-Mann-Whitney 191 test with a two-sided significance level of α=0.05 and a power of 1β = 0.8, a sample 192 size of 47 patients per group was required. With an estimated drop-out rate of 15-193 20% after randomization, 110 randomized patients were planned and 132 patients 194 were recruited to account for possible withdrawals prior to the start of the study. 195 196

  secondary target criteria were based on available data. 212 Treatment differences were tested by the use of the non-parametric Wilcoxon test for 213 continuous variables or by Fishers exact test for binary variables. All p-values are 214 two-sided. 215 Secondary efficacy variables included response based on a 50% rule of symptom 216 relief during the treatment (at least improvement in two out of four weeks within the 217 treatment period and improvement defined as at least one point reduction from 218 baseline). 219 All statistical analyses were perfomed using SAS version 9.1.3 for windows, SAS 220 Institute Inc., Cary, NC. 132 patients were included into the study and 122 patients were 226 successfully randomized to receive either placebo (N=62) or B. bifidum MIMBb75 227 (N=60). All randomized patients were analyzed for intent to treat (N=122). One 228 patient with no post randomization visit was excluded from the analysis of adverse 229 events. A total of 103 patients (49 placebo, 54 B. bifidum MIMBb75) were examined 230 as per protocol (Fig. 2

  incomplete evacuation" -on a seven-point Likert scale. B. bifidum MIMBb75 showed 259 a significant reduction of pain/discomfort by 260 -0.82 points [95%CI: -1.01; -0.63] vs. -0.18 [95%CI: -0.35; -0,01] in the placebo group 261 (p<0.0001), and distension/bloating by -0.92 points [95%CI: -1.15; -0.69] vs. -0.21 262 [95%CI: -0.37; -0.05] in the placebo group (p<0.0001) during the treatment. The 263 the wash-out phase. Urgency was significantly reduced by 264 -0.67 points [95%CI: -0,86; -0,48] vs. -0.21 [95%CI: -0,35; -0.07] in the placebo group 265

Figures 569 570 Fig. 1 .Fig. 2 .Fig. 3 .Fig. 5 .Fig. 6 .Fig. 7 .Fig. 8 .

 5701235678 Figures

Fig

  Fig.2.

Fig. 5

 5 Fig.5

  Fig.6

  Fig.8

F

  2% as alternators (61.7% in the bifidobacteria group, 54.8% in the placebo group) 238 with no significant differences between the bifidobacteria and the placebo group. 239 Demographics were well balanced between the treatment groups with about 67% 240 female patients and mean weight of 71 kg corresponding to a BMI of 24. The 241 average age of patients was 41 years in the placebo group and 37 years in the 242The primary endpoint was the reduction of the SGA of IBS symptoms on the 246 subject's global assessment diary. B. bifidum MIMBb75 significantly improved global 247 IBS symptoms by -0.88 points [95%CI: -1.07; -0.69] (from 2.95 in the run in phase to 248

	243	58.bifidobacteria group (Table 1).
	244
	245	Subject's global assessment (SGA) of IBS symptoms
		F o r
		P
		e
		e r
		R
		e
		v i e
		w
	235	in the
	236	bifidobacteria group, 19.4% in the placebo group), 19.7% as constipation-
	237	predominant IBS (15% in the bifidobacteria group, 24.2% in the placebo group) and
		10

Tables 516 Table 1 :

 5161 Demographic characteristics of the ITT-population 517 518

Table 2 :

 2 Summary of reported adverse events with suspected relationship by system 519

	520	organ class (SOC)
	521

Table 3 :

 3 Incidence of adverse events at visit 3-4 by preferred term (occurrence of 522

	523	more than 2 %)
	524
	525
	526
	527
	528
	529
	530
	531
	532
	533
	534
	535
	536
	537
	538
	539
	540
	541

did not come to visit 2, with no reason)

	CONSORT 2010 Flow Diagram
	Enrollment	Assessed for eligibility (n=132)
			Excluded (n=10)
			♦ Not meeting inclusion criteria (n=10)
	o	Randomized (n=122)
	r
	P
	e e r	Allocated to intervention (verum, n=60) ♦ Did not receive allocated intervention (give reasons) (n=0)
	Lost to follow-up (give reasons) (n=1, violation	R e i e w Lost to follow-up (give reasons) (n=1, Discontinued intervention (give reasons) (n=0) v
		Analysis
	Analysed (n= 60)		Analysed (n=59)
	♦ Excluded from analysis (give reasons) (n=0)		♦ Excluded from analysis (give reasons) (n=0)

♦ Received allocated intervention (n=60)

of exclusion criteria - antidepressive therapy-did not come to visit 2) Discontinued intervention (n=1, withdrawn at visit 2, no study product dispensed due to infection with antibiotic treatment)

  Allocation assessing outcomes) and how 11b If relevant, description of the similarity of interventions / 12a Statistical methods used to compare groups for primary and secondary outcomes 7, 8 Statistical methods 12b Methods for additional analyses, such as subgroup analyses and adjusted analyses 8, 9Results13a For each group, the numbers of participants who were randomly assigned, received intended treatment, and were analysed for the primary outcome , number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups / 17a For each primary and secondary outcome, results for each group, and the estimated effect size and its precision (such as 95% confidence interval) We strongly recommend reading this statement in conjunction with the CONSORT 2010 Explanation and Elaboration for important clarifications on all the items. If relevant, we also recommend reading CONSORT extensions for cluster randomised trials, non-inferiority and equivalence trials, non-pharmacological treatments, herbal interventions, and pragmatic trials.Additional extensions are forthcoming: for those and for up to date references relevant to this checklist, see www.consort-statement.org.

	Participant flow (a			9
	diagram is strongly
	recommended)	13b For each group, losses and exclusions after randomisation, together with reasons	CONSORT
				Flow Diagram
	Recruitment	14a Dates defining the periods of recruitment and follow-up	4, 5
		14b Why the trial ended or was stopped	/
	Baseline data	15	A table showing baseline demographic and clinical characteristics for each group	20
	Numbers analysed Outcomes and	16	For each group/
	estimation
		17b For binary outcomes, presentation of both absolute and relative effect sizes is recommended	Fig. 1-8
	Ancillary analyses	18	Results of any other analyses performed, including subgroup analyses and adjusted analyses, distinguishing	/
			pre-specified from exploratory
	Harms	19	All important harms or unintended effects in each group (for specific guidance see CONSORT for harms)	13
	Allocated to intervention (placebo, n=62) Discussion
	Limitations	20	Trial limitations, addressing sources of potential bias, imprecision, and, if relevant, multiplicity of analyses	15
	Generalisability	21	Generalisability (external validity, applicability) of the trial findings	13, 14
	Interpretation	22	Interpretation consistent with results, balancing benefits and harms, and considering other relevant evidence	13-15
	Other information
	Registration	23	Registration number and name of trial registry	/
	Protocol	24	Follow-Up Where the full trial protocol can be accessed, if available	/
	Funding	25	Sources of funding and other support (such as supply of drugs), role of funders	18

♦ Received allocated intervention (n=62)

♦ Did not receive allocated intervention (give reasons) (n=0) *

Alimentary Pharmacology & Therapeutic

CONSORT 2010 checklist of information to include when reporting a randomised trial*


Eugenio Bianchi

Daniele Regoli

Carlo Rovelli

The face amplitude of spinfoam quantum gravity

come

I. INTRODUCTION

A spinfoam sum over a given two-complex σ, formed by faces f joining along edges e in turn meeting at vertices v, is defined by the expression

Z σ = j f ,ie f d j f v A v (j f , i e ), (1)
where A v (j f , i e ) is the "vertex amplitude" and d j f is the "face amplitude". The sum is over an assignment j f of an irreducible representation of a compact group G to each face f and of an intertwiner i e to each edge e of the two-complex. The expression (1) is often viewed as a possible foundation for a background independent quantum theory of gravity [START_REF] Perez | Spin foam models for quantum gravity[END_REF]. In particular, a vertex amplitude A v (j f , i e ) that might define a quantum theory of gravity has been developed in [START_REF] Barrett | Relativistic spin networks and quantum gravity[END_REF][START_REF] Engle | The loop-quantum-gravity vertex-amplitude[END_REF][START_REF] Engle | Flipped spinfoam vertex and loop gravity[END_REF][START_REF] Pereira | Lorentzian LQG vertex amplitude[END_REF][START_REF] Engle | LQG vertex with finite Immirzi parameter[END_REF][START_REF] Livine | A new spinfoam vertex for quantum gravity[END_REF][START_REF] Freidel | A New Spin Foam Model for 4d Gravity[END_REF][START_REF] Kaminski | Spin-Foams for All Loop Quantum Gravity[END_REF] and is today under intense investigation (see [START_REF] Rovelli | A new look at loop quantum gravity[END_REF]). But what about the "measure factor" given by the face amplitude d j f ? What determines it?

The uncertainty in determining the face amplitude has been repeatedly remarked [START_REF] Pereira | Spin foams from simplicial geometry[END_REF][START_REF] Perez | A spin foam model without bubble divergences[END_REF][START_REF] Perez | Finiteness of a spinfoam model for Euclidean quantum general relativity[END_REF][START_REF] Crane | Perturbative finiteness in spin-foam quantum gravity[END_REF][START_REF] Perini | Self-energy and vertex radiative corrections in LQG[END_REF][START_REF] Bojowald | Spin foam quantization and anomalies[END_REF]. One way of fixing the face amplitude which can be found in the literature, for example, is to derive the sum [START_REF] Perez | Spin foam models for quantum gravity[END_REF] for general relativity (GR) starting from the analogous sum for a topological BF theory, and then implementing the constraints that reduce BF to GR as suitable constraints on the states summed over. For instance, in the Euclidean context GR is a constrained SO(4) BF theory. The state sum (1) is well understood for SO(4) BF theory: its face amplitude is the dimension of the SO(4) representation (j + , j -). The simplicity constraint fixes this to be of the form j ± = γ ± j f where γ ± = 1±γ 2 and γ is the Barbero-Immirzi parameter, and therefore

d j f = (2j + + 1)(2j -+ 1) = (2γ + j f + 1)(2γ -j f + 1). (2)
However, doubts can be raised against this argument. For instance, Alexandrov [START_REF] Alexandrov | The new vertices and canonical quantization[END_REF] has stressed the fact that the implementation of second class constraints into a Feynman path integral in general requires a modification of the measure, and here the face amplitude plays precisely the role of such measure, since A v ∼ e i Action . Do we have an independent way of fixing the face amplitude?

Here we argue that the face amplitude is uniquely determined for any spinfoam sum of the form (1) by three inputs: (a) the choice of the boundary Hilbert space, (b) the requirement that the composition law holds when gluing two-complexes; and (c) a particular "locality" requirement, or, more precisely, a requirement on the local composition of group elements.

We argue below that these requirements are implemented if Z is given by the expression

Z σ = dU v f v A v (U v f ) f δ(U v1 f ...U v k f ), (3)
where

U v f ∈ G, v 1 .
..v k are the vertices surrounding the face f , and A v (U v f ) is the vertex amplitude A v (j f , i e ) expressed in the group element basis [START_REF] Bianchi | Spinfoams in the holomorphic representation[END_REF]. Then we show that this expression leads directly to (1), with arbitrary vertex amplitude, but a fixed choice of face amplitude, which turns out to be the dimension of the representation j of the group G,

d j = dim(j). (4)
In particular, for quantum gravity this implies that the BF face amplitude (2) is ruled out, and should be replaced (both in the Euclidean and in the Lorentzian case) by the SU (2) dimension

d j = 2j + 1. (5)
Equation ( 3) is the key expression of this paper; we begin by showing that SO(4) BF theory (the prototypical spinfoam model) can be expressed in this form (Section II). Then we discuss the three requirements above and we show that (3) implements these requirements. (Section III). Finally we show that (3) gives (1) with the face amplitude (4) (Section IV).

The problem of fixing the face amplitude has been discussed also by Bojowald and Perez in [START_REF] Bojowald | Spin foam quantization and anomalies[END_REF]. Bojowald and Perez demand that the amplitude be invariant under suitable refinements of the two-complex. This request is strictly related to the composition law that we consider here, and the results we obtain are consistent with those of [START_REF] Bojowald | Spin foam quantization and anomalies[END_REF].

v 1 v 2 v 3 v k . . . U v1 f h -1 v1ek h v1e1 h -1 v2e1 f U e1 FIG. 1: Schematic definition of the group elements hve, U v f
and Ue associated to a portion of a face f of the two-complex.

II. BF THEORY

It is well known that the partition function [START_REF] Perez | Spin foam models for quantum gravity[END_REF] for BF theory can be rewritten in the form (see [START_REF] Perez | Spin foam models for quantum gravity[END_REF])

Z σ = dU e f δ(U e1 ...U e k ), (6)
where U e are group elements associated to the oriented edges of σ, and (e 1 , ..., e k ) are the edges that surround the face f . Let us introduce group elements h ve , labelled by a vertex v and an adjacent edge e, such that

U e = h ve h -1 v e (7)
where v and v are the source and the target of the edge e (see Figure 1). Then we can trivially rewrite [START_REF] Engle | LQG vertex with finite Immirzi parameter[END_REF] as

Z σ = dh ve f δ (h v1e1 h -1 v2e1 ) ... (h v k e k h -1 v1e k ) . ( 8
)
Now define the group elements

U v f = h -1 ve h ve (9)
associated to a single vertex v and two edges e and e that emerge from v and bound the face f (see Figure 1). Using these, we can rewrite [START_REF] Engle | LQG vertex with finite Immirzi parameter[END_REF] as

Z σ = dh ve dU v f v,f v δ(U v f , h -1 ve h ve ) f δ(U v1 f ...U v k f ),
where the first product is over faces f v that belong to the vertx v, and then a product over all the vertices of the two-complex. Notice that this expression has precisely the form (3), where the vertex amplitude is

A v (U v f ) = dh ve f v δ(U v f , h ve h -1 ve ), (11)
which is the well-known expression of the 15j Wigner symbol (the vertex amplitude of BF in the spin network basis) in the basis of the group elements.

We have shown that the BF theory spinfoam amplitude can be put in the form [START_REF] Engle | The loop-quantum-gravity vertex-amplitude[END_REF]. We shall now argue that (3) is the general form of a local spinfoam model that obeys the composition law.

III. THREE INPUTS

(a) Hilbert space structure. Equation ( 1) is a coded expression to define the amplitudes

W σ (j l , i n ) = j f ,ie f d j f v A v (j f , i e ; j l , i n ), (12)
defined for a two-complex σ with boundary, where the boundary graph Γ = ∂σ if formed by links l and nodes n. The spins j l are associated to the links l, as well as to the faces f that are bounded by l; the intertwiners i n are associated to the nodes n, as well as to the edges e that are bounded by n. The amplitude of the vertices that are adjacent to these boundary faces and edges depend also on the external variables (j l , i n ).

In a quantum theory, the amplitude W (j l , i n ) must be interpreted as a (covariant) vector in a space H Γ of quantum states. 1 We assume that this space has a Hilbert space structure, which we know. In particular, we assume that

H Γ = L 2 [G L , dU l ] ( 13
)
where L is the number of links in Γ and dU l is the Haar measure. Thus we can interpret [START_REF] Perez | A spin foam model without bubble divergences[END_REF] as

W σ (j l , i n ) = j l , i n | W ( 14
)
where |j l , i n is the spin network function

U l | j l , i n = ψ j l ,in (U l ) = l R j l (U l ) • n i n . ( 15
)
Here R j (U l ) are the representation matrices in the representation j and the i form an orthonormal basis in the intertwiner space. See for instance [START_REF] Rovelli | A new look at loop quantum gravity[END_REF][START_REF] Rovelli | Quantum Gravity[END_REF] for details.

Using the scalar product defined by ( 13), we have

j l , i n | j l , i n = dU l ψ j l ,in (U l )ψ j l ,i n (U l ) = l dim(j l ) δ j l j l n δ ini n . ( 16
)
where dim(j) is the dimension of the representation j. Therefore the spin-network functions ψ j l ,in (U l ) are not

v 1 v 2 v k U v1 f U vk f U v2 f U l l l . . . f FIG. 2:
Cutting of a face of the two-complex. The holonomy U l is attached to a link of the boundary spin-network and satisfies equation (23).

normalized. (These dim(j) normalization factors are due to the convention chosen: they have nothing to do with the dimension of the representation that appears in [START_REF] Engle | Flipped spinfoam vertex and loop gravity[END_REF].)

The resolution of the identity in this basis is

1 1 = j l ,in l dim(j l ) |j l , i n j l , i n |. ( 17
)
(b) Composition law. In non relativistic quantum mechanics, if U (t 1 , t 0 ) is the evolution operator from time t 0 to time t 1 , the composition law reads

U (t 2 , t 0 ) = U (t 2 , t 1 )U (t 1 , t 0 ). (18)
That is, if |n is an orthonormal basis,

f |U (t 2 , t 0 )| i = n f |U (t 2 , t 1 )| n n |U (t 1 , t 0 )| i .
Let us write an analogous condition of the spinfoam sum. Consider for simplicity a two-complex σ = σ 1 ∪ σ 2 without boundary, obtained by gluing two two-complexes σ 1 and σ 2 along their common boundary Γ. Then we require that W satisfies the composition law

Z σ1∪σ2 = W σ2 | W σ1 , ( 20
)
as discussed by Atiyah in [START_REF] Atiyah | Topological Quantum Field Theories[END_REF]. Notice that to formulate this condition we need the Hilbert space structure in the space of the boundary states.

(c) Locality. As a vector in H Γ , the amplitude W (j l , i n ) can be expressed on the group element basis

W (U l ) = U l | W (21) = j l ,in l dim(j l ) ψ j l ,in (U l )W (j l , i n ).
Similarly, the vertex amplitude can be expanded in the group element basis

A v (U v f ) = U v f | A v (22) = j v f ,i v n f v dim(j v f ) ψ j v f ,i v n (U v f )A v (j v f , i v n ).
Notice that here the group element U v f and the spin j v f are associated to a vertex v and a face f adjacent to v.

Similarly, the intertwiner i v n is associated to a vertex v and a node n adjacent to v. Consider a boundary link l that bounds a face f (see Figure 2). Let v 1 ...v k be the vertices that are adjacent to this face. We say that the model is local if the relation between the boundary group element U l and the vertices group elements U v f is given by

U l = U v1 f ... U v k f . ( 23
)
In other words: if the boundary group element is simply the product of the group elements around the face.

Notice that a spinfoam model defined by ( 3) is local and satisfies composition law in the sense above. In fact, (3) generalizes immediately to

W σ (U l ) = dU v f v A v (U v f ) internal f δ(U v1 f ...U v k f ) × external f δ(U v1 f ...U v k f U -1 l ). ( 24
)
Here the first product over f is over the ("internal") faces that do not have an external boundary; while the second is over the ("external") faces f that are also bounded by the vertices v 1 , ..., v k and by the the link l. It is immediate to see that locality is implemented, since the second delta enforces the locality condition (23). Furthermore, when gluing two amplitudes along a common boundary we have immediately that

dU l W σ1 (U l ) W σ2 (U l ) = Z σ1∪σ2 (25)
because the two delta functions containing U l collapse into a single delta function associated to the face l, which becomes internal. Thus, (3) is a general form of the amplitude where these conditions hold.

In [START_REF] Bojowald | Spin foam quantization and anomalies[END_REF], Bojowald and Perez have considered the possibility of fixing the face amplitude by requiring the amplitude of a given spin/intertwiner configuration to be equal to the amplitude of the same spin/intertwiner configuration on a finer two-simplex where additional faces carry the trivial representation. This requirement imply essentially that the amplitude does not change by splitting a face into two faces. It is easy to see that (3) satisfies this condition. Therefore (3) satisfies also the Bojowald-Perez condition.

IV. FACE AMPLITUDE

Finally, let us show that (3) implies ( 1) and (4). To this purpose, it is sufficient to insert (22) into (3). This gives

Z σ = dU v f v j v f ,i v n f v dim(j v f ) ψ j v f ,i v n (U v f )A v (j v f , i v n ) × f δ(U v1 f ...U v k f ). ( 26
)
Expand then the delta function in a sum over characters

Z σ = dU v f v j v f ,i v n f v dim(j v f ) ψ j v f ,i v n (U v f )A v (j v f , i v n ) × f j f dim(j f ) Tr(R j f (U v1 f ) • • • R j f (U v k f )).
We can now perform the group integrals. Each U v f appears precisely twice in the integral: once in the sum over j v f and the other in the sum over j f . Each integration gives a delta function δ j v f ,j f , which can be used to kill the sum over j v f dropping the v subscript. Following the contraction path of the indices, it is easy to see that these contract the two intertwiners at the opposite side of each edge. Since intertwiners are orthonormal, this gives a delta function δ i v n ,i v n which reduces the sums over intertwiners to a single sum over i n := i v n = i v n . Bringing everything together, and noticing that the dim(j) factor from the group integrations cancels the one in the integral, we have

Z σ = j f in f dim(j f ) v A v (j v f , i v n ). (27)
This is precisely equation (1), with the face amplitude given by (4). Notice that the face amplitude is well defined, in the sense that it cannot be absorbed into the vertex amplitude (as any edge amplitude can). The reason is that any factor in the vertex amplitude depending on the spin of the face contributes to the total amplitude at a power k,

where k is the number of sides of the face. The face amplitude, instead, is a contribution to the total amplitude that does not depend on k. This is also the reason why the normalization chosen for the spinfoam basis does not affect the present discussion: it affects the expression for the vertex amplitude, not that for the face amplitude.

By an analogous calculation one can show that the same result holds for the amplitudes W : equation [START_REF] Perez | A spin foam model without bubble divergences[END_REF] follows from (24) expanded on a spin network basis.

In conclusion, we have shown that the general form (3) of the partition function, which implements locality and the composition law, implies that the face amplitude of the spinfoam model is given by the dimension of the representation of the group G which appears in the boundary scalar product [START_REF] Perez | Finiteness of a spinfoam model for Euclidean quantum general relativity[END_REF].

In general relativity, in both the Euclidean and the Lorentzian cases, the boundary space is

H Γ = L 2 [SU (2) L , dU l ], (28)
therefore the face amplitude is d j = dim SU(2) (j) = 2j +1, and not the SO( 4) dimension (2), as previously supposed.

Notice that such d j = 2j + 1 amplitude defines a theory that is far less divergent than the theory defined by [START_REF] Barrett | Relativistic spin networks and quantum gravity[END_REF]. In fact, the potential divergence of a bubble is suppressed by a power of j with respect to [START_REF] Barrett | Relativistic spin networks and quantum gravity[END_REF]. In [START_REF] Perini | Self-energy and vertex radiative corrections in LQG[END_REF], it has been shown that the d j = 2j + 1 face amplitude yields a finite main radiative correction to a five-valent vertex if all external legs set to zero.

If Γ has two disconnected components interpreted as "in" and an "out" spaces, then H Γ can be identified as the tensor product of the "in" and an "out" spaces of non-relativistic quantum mechanics. In the general case, H Γ is the boundary quantum state in the sense of the boundary formulation of quantum theory [

[START_REF] Oeckl | A 'general boundary' formulation for quantum mechanics and quantum gravity[END_REF][START_REF] Rovelli | Quantum Gravity[END_REF].

Acknowledgements

We thank Elena Magliaro, Antonino Marcianò, Claudio Perini, and Simone Speziale for useful discussions. EB gratefully acknowledges funding for this work through a Marie Curie Fellowship of the European Union.