Allison Muller
email: a3muller@chu-besancon.fr

K Romand

D Suplisson

J F Nattero

J Leroy

X Bertrand

Impact of a long-term antibiotic stewardship program targeting fluoroquinolones in a French local hospital Impact à long terme d'une politique d'épargne des fluoroquinolones dans un hôpital local français

Keywords: antibiotic stewardship program, fluoroquinolones, bacterial resistance bon usage des antibiotiques, fluoroquinolones, résistance bactérienne

HAL is

Introduction

Fluoroquinolones (FQs) are a major antibiotic class in human health. They are recommended in the treatment of severe infections such as bone and joint infections, prostatitis, or pulmonary infections (e.g., legionellosis). Their wide spectrum of activity, rapid bactericidal effect, excellent oral absorption, and tolerability make them easy-to-prescribe antibiotics and has resulted in their extensive use. However, at equal consumption, the ecological impact of FQs seems to be more important than that of other antibiotic classes; notably, the worldwide expansion of the major clones Escherichia coli ST131 H30-Rx, methicillin-resistant Staphylococcus aureus EMRSA-15, and Clostridium difficile 027 are caused by the clinical use of FQs [START_REF] Zakour | Sequential Acquisition of Virulence and Fluoroquinolone Resistance Has Shaped the Evolution of Escherichia coli ST131[END_REF][START_REF] Stoesser | Evolutionary History of the Global Emergence of the Escherichia coli Epidemic Clone ST131[END_REF][START_REF] Holden | A genomic portrait of the emergence, evolution, and global spread of a methicillin-resistant Staphylococcus aureus pandemic[END_REF][START_REF] Warny | Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe[END_REF][START_REF] Kuijper | the ESCMID Study Group for Clostridium difficile (ESGCD), EU Member States and the European Centre for Disease Prevention and Control (ECDC)[END_REF]. Alternatives to FQ prescription are available in numerous clinical situations and antibiotics with a lesser ecological impact may be substituted to FQs to preserve their activity for complicated hospital-acquired infections. An audit was conducted in our region in 2006 to evaluate the adequacy and relevance of FQ prescriptions. Results highlighted an important misuse of this antibiotic class [START_REF] Leroy | Audit régional de l'usage des fluoroquinolones à l'hôpital et en ville : y a-t-il une surconsommation de ces antibiotiques ?[END_REF]. The local hospital of Morteau participated in the regional audit and decided to take part in an antibiotic stewardship program targeting FQs.

We aimed to assess the efficiency of the antibiotic stewardship program on FQ consumption and its impact on resistance rates. The secondary objective was to determine whether the antibiotic stewardship program had an impact on FQ consumption in the community setting.

Physicians who prescribed in the hospital were family physicians (FPs) and also practiced in the community.

Methods

We conducted this observational study in the local hospital of Morteau, a 166-bed facility with a health sector (70 beds: 20 acute care, 20 post-acute care and rehabilitation, and 30 long-term care beds) and a medico-social sector (96 nursing home beds). A medical home is also affiliated with the hospital. Seventeen family physicians, who all have outpatient and inpatient consultations, work in the facility and a pharmacist works full-time in the hospital pharmacy. The Morteau hospital is located in the Franche-Comté region, in the Val de Morteau (eastern France; 19,550 inhabitants in 2012) [START_REF]Comparateur de territoire Intercommunalité-Métropole de CC du Val de Morteau[END_REF]. There are three community pharmacies in the city of Morteau.

Design and setting

Regional guidelines drafted by a multidisciplinary group of specialists were developed to improve the prescription of FQs. A regional audit evaluating the prescribers' compliance with guidelines in hospital and community settings was then conducted in the Franche-Comté region in 2006 [START_REF] Leroy | Audit régional de l'usage des fluoroquinolones à l'hôpital et en ville : y a-t-il une surconsommation de ces antibiotiques ?[END_REF]. Professional practices were evaluated before the beginning of the prescriber training program. Results of the audit highlighted a misuse of FQs in the hospital of Morteau as none of the evaluated prescriptions complied with regional guidelines. Local guidelines on empirical treatment regimens were released in 2007. The aim of the antibiotic stewardship program was to reduce the rate of unnecessary prescriptions of FQs. Two main messages were for instance included in the guidelines for UTIs: (i) a reminder on the proper prescription of the cytobacteriological examination of urine; (ii) FQs should not be used for uncomplicated acute cystitis; fosfomycin-trometamol (single dose of 3 g) or nitrofurantoin (300 mg/day for 5 days) should be the preferred first-line treatment. The working group (including the president of the medical committee, the medico-social sector coordinating physician, the regional infectious diseases specialist, the pharmacist, and the biologist) defines the policy for an appropriate use of antibiotics, meets several times a year, organizes an annual professional practices assessment, and suggests updates to the local guidelines.

The following monitoring indicators were set up in the hospital setting: total consumption of antibiotics (ATC code J01) and of FQs expressed in defined daily doses (DDD) per 1,000 patient-days, and surveillance of the nalidixic acid resistance among Escherichia coli strains.

Bacteriological data was supplied by the laboratory biologist who analyzes the samples of the patients of Morteau hospital, and the resistance of E. coli strains was evaluated by antimicrobial susceptibility testing. The use of FQs in the community was assessed by the number of packs sold in one community pharmacy which provided its sales data.

Results

Hospital setting

An almost 10-fold decrease in hospital FQ consumption was observed between 2006 and 2015 (from 41.1 to 5.9 DDD/1,000 patient-day). The most important reduction was observed during the first year of the antibiotic stewardship program (-66.9%). We observed a delayed effect on the resistance to nalidixic acid among E. coli strains, which started decreasing in 2011 (716 strains were analyzed during the study period) (Figure 1).

The fosfomycin consumption increased but remained very low, and the nitrofurantoin consumption decreased after initially increasing until 2008 (data not shown). The resistance of E. coli strains to these antibiotics remained low (<15%) and even decreased for nitrofurantoin. The consumption of most antibiotic classes was stable during the study period, especially that of third-generation cephalosporins (3GC) which has even decreased.

The consumption of broad-spectrum antibiotics remained close to zero (piperacillintazobactam, carbapenems, aminoglycosides, glycopeptides). An increased consumption of amoxicillin and amoxicillin-clavulanic acid was observed in 2010, followed by a decrease in the following years (data not shown). The global consumption of antibiotics decreased, except in 2010 which was marked by a peak in consumption (Figure 2). Data concerning the health sector and the medico-social sector was analyzed separately for the years 2008 to 2015 (data not available for 2006 and 2007): a 14.7% decrease in FQ consumption and a 20.3% increase in the global antibiotic consumption were observed for the health sector, whereas in the medico-social sector the FQ consumption remained stable and the global antibiotic consumption decreased by 18.2% (data not shown for FQ consumption) (Figure 2).

Community setting

The consumption of norfloxacin in the community setting decreased by 88% during the study period. A marked reduction was also observed in the consumption of lomefloxacin (-54%).

The consumption of ofloxacin remained low and steady over the study period (Figure 3).

The consumption of fosfomycin and nitrofurantoin concomitantly increased (respectively +68% and +45.7% from 2007 to 2013) (Figure 3).

Discussion

The cautious use of antibiotics in hospital and community settings has become a public health priority. Numerous studies assessing antimicrobial prescription in hospitals all around the world highlighted inappropriate practices and high levels of use, which led to a worrying increase in bacterial resistance. The implementation of antibiotic stewardship programs such as the diffusion of guidelines or surveys on antibiotic consumptions, are not systematically associated with an improved use of antibiotics. Antibiotic consumptions are still high, despite improvement measures, in particular in France which is one of the biggest antibiotic consumers in Europe [8]. This issue is particularly concerning for FQs as their misuse has an important ecological impact.

This study showed that the implementation of an antibiotic stewardship program focused on FQs in a local hospital can be effective in reducing the use of FQs in the long-term. We observed a sharp decrease (-85%) in FQ consumption between 2006 and 2015 at the Morteau hospital; the hospital used to be a high FQ consumer and is now a small consumer [START_REF]Observatoire de la consommation antibiotique et de la résistance bactérienne -OSCAR[END_REF]. Compared with the 2015 national results from the French national network [10], the FQ consumption in the health sector of the Morteau hospital was lower than the median FQ consumption for comparable health care facilities (hospitals with ≤33% of short stay beds) (12.8 vs 23.9 DDD per 1,000 patient-days, respectively).

The main target of the antibiotic stewardship program was FQs prescribed for UTIs. Our results suggest that the decreased FQ prescription was mainly associated with a decrease in the use of cytobacteriological examinations of urine (-62% between 2008 and 2016). Indeed, we did not observe any compensatory increase in nitrofurantoin and fosfomycin consumptions. Moreover, the antibiotic prescription policy for UTI treatment had to be updated in 2011 because of the concern of family physicians about the adverse events of nitrofurantoin. These concerns prompted family physicians to come back to prescribing FQs instead of nitrofurantoin, which presumably accounts for the FQ consumption peak observed that year [START_REF] Slekovec | When the precautionary principle disrupts 3 years of antibiotic stewardship: nitrofurantoin in the treatment of urinary tract infections[END_REF].

During the study period the overall antibiotic consumption in the whole hospital decreased, unlike national results [10]. The decrease in FQ use was not balanced by an increased use of other antibiotic classes. We therefore did not observe the so-called "squeezing the antibiotic balloon" effect, i.e. when the decreased use of an antibiotic class leads to the increased use of another one, by way of compensation [START_REF] Peterson | Squeezing the antibiotic balloon: the impact of antimicrobial classes on emerging resistance[END_REF].

A sharp decrease in nalidixic acid resistance among E. coli strains was observed after the decrease in FQ consumption (-57.2% between 2006 and 2015), although after a long period of time. The absence of immediate decrease in FQ resistance among E. coli strains after such a decrease in FQ use was also observed by Langford in a Canadian hospital [START_REF] Langford | Antimicrobial Stewardship in the Microbiology Laboratory: Impact of Selective Susceptibility Reporting on Ciprofloxacin Utilization and Susceptibility of Gram-Negative Isolates to Ciprofloxacin in a Hospital Setting[END_REF]. Several studies showed that the carriage of multidrug-resistant E. coli may be prolonged even in the absence of antimicrobial selection pressure [START_REF] Bar-Yoseph | Natural history and decolonization strategies for ESBL/carbapenem-resistant Enterobacteriaceae carriage: systematic review and meta-analysis[END_REF], particularly in nursing homes [START_REF] Overdevest | Prolonged colonisation with Escherichia coli O25:ST131 versus other extendedspectrum beta-lactamase-producing E. coli in a long-term care facility with high endemic level of rectal colonisation, the Netherlands, 2013 to 2014[END_REF]. We may thus assume that the time lag observed between the reduction in FQ use and the effect on FQ resistance is associated with this prolonged carriage of multidrug-resistant E. coli strains in patients hospitalized in long-term care facilities. It will take several years to achieve a sufficient patient turnover and to observe the effect of FQ use reduction. Although this hypothesis has to be confirmed, it suggests that in specific situations the effect of an antibiotic stewardship program may be observed years after its implementation.

All physicians of this study who work in the local hospital are family physicians who also have outpatient consultations in the medical home affiliated with the hospital. The positive impact of the guidelines could also impact the prescriptions for these ambulatory patients as a sharp decrease was observed in the number of FQ packs sold in a community pharmacy of Morteau, especially for the norfloxacin sales, whereas an increase was observed in fosfomycin and nitrofurantoin prescriptions. However, no evidence proves the causal relation between the antibiotic stewardship program implemented in the Morteau hospital and the decrease in FQ sales in the community pharmacy.

A key component of this improvement in practices is the deep involvement of the physicians, who regularly take part in the anti-infective agent committee meetings, but also of the hospital pharmacist who monitors and validates antibiotic prescriptions on a daily basis: when non-compliance is observed, a notice is sent to the prescriber via the computerized medical record, and every non-compliant prescription is collected by the pharmacist to be presented in the next anti-infective agent committee meeting. The involvement of an infectious disease specialist also impacted antibiotic prescription practices, in particular in local facilities where only family physicians who are not always aware of the latest recommendations work. Another key component of this measure is the continuous learning opportunity for the prescribers during the meetings with the pharmacist and the infectious disease specialist. Subjects discussed are chosen according to non-compliance behaviors targeted by the professional practice evaluation.

This study has several limitations: first, community data is scarce as data about FQ sales in community pharmacies is available for only one of the three pharmacies in the city, and the non-availability of data on bacterial resistance in the community prevents the evaluation of the effect of FQ prescription decrease in the community. Another limitation is that data on bacterial resistance at the hospital is based on a small number of strains isolated from hospitalized patients (716 strains between 2007 and 2015); the resistance rate should therefore be interpreted with caution. This data also included patients of the whole facility and was not stratified between patients of the health sector and of the medico-social sector.

Conclusion

Our study demonstrated that an antimicrobial stewardship program in a local hospital can greatly reduce the use of FQs, decrease FQ resistance, and may have a positive impact on the surrounding community.

Figure 1 .Figure 1 .

 11 Figure 1. Trends of fluoroquinolone consumption (DDD/1,000 patients-days) and resistance to nalidixic acid among E. coli strains (% of resistant strains) for the whole facility between 2006 and 2015

Figure 2 .

 2 Figure 2. Changes in the global antibiotic consumption (DDD/1,000 patient-days) for the whole facility and for the health and medico-social sectors (data missing for 2006 and 2007) between 2006 and 2015

Figure 2 .Figure 3 .Figure 3 .

 233 Figure 2. Évolution des consommations d'antibiotiques globales (DDJ/1 000 JH) pour l'ensemble de l'établissement et pour les secteurs sanitaire et médico-social (données manquantes pour 2006 et 2007) entre 2006 et 2015

Funding

None

Declaration of interests

The authors declare no competing interests.

Contribution of authors


Prof Fabrice Bartolomei
email: fabrice.bartolomei@ap-hm.fr

Stanislas Lagarde

D Scavarda

R Carron

C G Bénar

F Picard

The role of the dorsal anterior insula in ecstatic sensation revealed by direct electrical brain stimulation

Abstract

Background: An ecstatic phenomenon is an altered state of consciousness with a sense of "hyper-reality", and a complete present-moment awareness with a feeling of union with the Universe. A better understanding of the network mechanisms underlying this fascinating subjective experience may help to unravel some mysteries of human consciousness. Insula has been recently proposed to be a key region to elicit these symptoms.

Objective/Hypothesis: We studied functional connectivity changes in several brain areas during the induction of ecstatic auras by direct electrical stimulation of the dorsal anterior insular cortex in patients with refractory focal epilepsy implanted with intracerebral electrodes (stereotactic-EEG, SEEG) in the context of their pre-surgical evaluation.

Methods: Three patients were selected on the basis of the occurrence of ecstatic symptoms triggered by direct intracerebral electrical stimulation (ES) of the antero-dorsal part of the insula. ES was performed (50 Hz, 1.5-2.1 mA, in a bipolar fashion to each contact in the gray matter during a 3 second period) to map functional cortices and trigger habitual seizures.

One stimulation inducing ecstatic changes in each patient was analyzed. Functional connectivity analysis was performed by measuring interdependencies (nonlinear regression analysis based on the h² coefficient) between SEEG signals before and after stimulations.

Results:

In all patients, only the stimulation of dorsal anterior insula was able to reproduce an ecstatic aura. We observed a significant increase of functional connectivity values between several brain regions in the immediate period following stimulations. The most commonly implicated region was the dorsal anterior insula. Out-degrees (a measure intended to identify leading structures in a network) identified the dorsal anterior insula as the most common leading region in the induced networks.

Conclusion(s): Our findings bring additional support in favor of a major role played by the dorsal anterior insula in ecstatic experiences.

Highlights

-In 3 patients, only the direct stimulation of the dorsal anterior insula induced ecstatic phenomenon.

Introduction

The epileptic "aura" is a subjective phenomenon that sometimes precedes the visible clinical features of a seizure and that is related to the beginning of seizures in the brain. A few patients with epilepsy experience a type of aura referred to as "ecstatic". Ecstatic epileptic auras consist of a sensation of "hyper-reality". The patients use terms such as "clarity", "evidence", "certainty", "understanding", "insight", "enlightenment" or "epiphany". Dostoevsky gave a famous description of such seizures, that he experienced himself and in which he reached a wonderful state of clarity and bliss [START_REF] Rayport | Dostoevsky's epilepsy: a new approach to retrospective diagnosis[END_REF]. The phenomenological detail of the ecstatic aura includes three important points: bliss ("the immense joy that feels me is above physical sensations" [START_REF] Gschwind | Ecstatic Epileptic Seizures: A Glimpse into the Multiple Roles of the Insula[END_REF]), a sense of certainty ("things suddenly seemed self-evident" [START_REF] Picard | Predictions, perception, and a sense of self[END_REF]), and a sense of timelessness or a sense of being in an eternal now ("These moments are without beginning and without end" [START_REF] Gschwind | Ecstatic Epileptic Seizures: A Glimpse into the Multiple Roles of the Insula[END_REF]). The feeling of certainty during ecstatic auras and a frequent feeling of oneness with the Universe are sometimes interpreted as mystical or religious by some individuals, depending on their previous religious beliefs, and may change their life forever.

The suspected location of the ecstatic ictal symptoms was the temporal lobe until recently, however evidence has converged more recently pointing to an involvement of the dorsal anterior insula probably within a larger neural network [START_REF] Gschwind | Ecstatic Epileptic Seizures: A Glimpse into the Multiple Roles of the Insula[END_REF][START_REF] Picard | Ecstatic epileptic seizures: a potential window on the neural basis for human self-awareness[END_REF][START_REF] Picard | State of belief, subjective certainty and bliss as a product of cortical dysfunction[END_REF]. We could support this hypothesis of insular involvement by the finding of a first patient in whom the direct stimulation of the antero-dorsal part of the insula induced her ecstatic auras [START_REF] Picard | Induction of a sense of bliss by electrical stimulation of the anterior insula[END_REF]. In the present report, we studied functional connectivity changes in several brain areas during the induction of ecstatic auras by electrical stimulation of the dorsal anterior insula in 3 patients having intracerebral electrodes for presurgical evaluation. Physiological functions in the brain require the functional interactions between multiple distinct neural networks. The measure of statistical interdependencies between signals of brain activity is an estimation of functional interactions and is referred to as 'functional connectivity" [START_REF] Bressler | Understanding cognition through large-scale cortical networks[END_REF]. The underlying assumption is that such correlations reflect, at least in part, functional interactions between different brain areas.

Materials and methods

Selection of patients

Subjects were 3 patients (P1, P2 and P3) (Table 1) with drug-resistant focal epilepsy, candidate to surgical treatment. They were studied in the Epilepsy Unit of the Timone Hospital, Marseille, France. For all these patients, intracerebral recordings with stereotactic EEG (SEEG) were performed in the context of their pre-surgical evaluation. These three patients were selected in the period 2011-2017 because they reported during intracerebral stimulation the occurrence of ecstatic symptoms defined by seizures with the feeling of (1) intense positive emotion (bliss), (2) enhanced physical well-being, and (3) heightened selfawareness or heightened perception of the external world (clarity) [START_REF] Gschwind | Ecstatic Epileptic Seizures: A Glimpse into the Multiple Roles of the Insula[END_REF]. These feelings were similar to those reported during their spontaneous auras. Patients with only vague pleasant phenomenon, with orgasmic/sexual feeling or with associated perceptual hallucinations (feeling of a presence, auditory hallucinations…) were not selected. Patients signed informed consent, and the study was approved by the Institutional Review board (IRB00003888) of INSERM. Patient data are summarized in Table 1.

SEEG recordings and electrical stimulation

SEEG recordings were made thanks to intracerebral electrodes with multiple contacts (10-15 contacts) as previously described [START_REF] Badier | Technical solutions for simultaneous MEG and SEEG recordings: towards routine clinical use[END_REF]. The electrodes were positioned in each patient based upon hypotheses about the localization of the epileptogenic zone, formulated from available noninvasive information. Each patient had a comprehensive evaluation including detailed history, neurological examination, neuropsychological testing, high resolution magnetic resonance imaging (MRI), and video-electroencephalography (EEG) recording of seizures before SEEG. Signals were recorded on a 256-channels Natus™ system. They were sampled at 1024 Hz. In total, 3 stimulations in 3 different patients, were selected for this study. Electric stimulation was produced by a regulated neurostimulator designed for safe diagnostic stimulation of the human brain (Inomed®). High frequency stimulation at 50 Hz (pulse duration 1 millisecond, intensity 0.5-2 mA) was applied in a bipolar fashion to each of the pairs of contacts in the gray matter during a 3-5 seconds period. During stimulation, patient was sitting in bed and was asked to read or count.

One stimulation inducing ecstatic changes in each patient was analyzed (table 1). In each patient we studied the interdependencies between bipolar SEEG signals (i.e., difference of contiguous contacts).

SEEG analysis: estimation of functional connectivity changes

Functional connectivity is defined as the statistical dependencies between two neurophysiological responses. In our case, these functional connectivity measures are directed, in the sense that we assessed nonlinear correlations as a function of time lag between electrodes.

Definition of the period of interest

For each stimulation we chose two periods of analysis. The background period was a period of 15 s immediately preceding the stimulation and is referred here as PreS ("pre stimulation").

The second period was a 10 s period immediately following the stimulation artifact and referred as PoS (post stimulation). All the analyses were performed with bipolar derivation (subtraction of consecutive channels). During stimulation, the bipolar contacts are disconnected and cannot be studied for connectivity analysis. Thus we have taken two adjacent contacts representative of the stimulated region. Signals were filtered in Anywave software [START_REF] Colombet | AnyWave: a cross-platform and modular software for visualizing and processing electrophysiological signals[END_REF] (software available at http://meg.univamu.fr/wiki/AnyWave), with Butterworth filters of order 4. We used only a 1Hz high pass filter in all the analyses.

Estimation of functional connectivity using Non-linear correlation (h²)

Signals were analyzed with the AnyWave software [START_REF] Colombet | AnyWave: a cross-platform and modular software for visualizing and processing electrophysiological signals[END_REF], which computes a nonlinear regression analysis based on the h² coefficient [START_REF] Pijn | Propagation of electrical activity: nonlinear associations and time delays between EEG signals[END_REF] . In summary, a piecewise linear regression is performed between each pair of signals, testing all the shifts of one signal relative to the other within a maximum lag. The h² is the coefficient of determination which measures the goodness of fit of the nonlinear regression -equivalent to the R-squared used in linear regression. The h² is bounded between 0 (no correlation) and 1 (maximal correlation) andcontrary to the R-squared -is not symmetric [START_REF] Wendling | Interpretation of interdependencies in epileptic signals using a macroscopic physiological model of EEG[END_REF]. We used a sliding window of 3 seconds with an overlap of 2 seconds, and a maximum delay between signals of 100 ms. For each pair of signals (X,Y), we retained the maximal value across delays and across directions (X to Y or Y to X). This value corresponded to a delay between signals, which was used to define the direction of the link. We thus obtained functional connectivity graphs, with each channel representing a node of the graph, and h 2 values the strength of the link between two nodes [START_REF] Courtens | Graph Measures of Node Strength for Characterizing Preictal Synchrony in Partial Epilepsy[END_REF].

Connectivity graphs were obtained for the three stimulations (one for each patient) between bipolar signals derived from the grey matter of 7 regions. We computed all pair-wise interactions from 7 regions (42 connections), and for each connectivity pair, we took the maximum (resulting in a final number of 21 interactions). For clinical reasons, the sampling of the brain could be variable from one patient to the other. Therefore we selected a common set of regions (amygdala, hippocampus, temporal neocortex, dorsal anterior insular cortex, orbitofrontal cortex, dorsolateral prefrontal cortex (DLPFC)) and another neocortical region that was variable from one patient to the other (see Fig 2 for the electrodes placement on 3D mesh of the MRI in the three patients).

Graph measures

We summarized the connectivity graphs with a graph measures, the node degrees. This entailed thresholding the graphs, and then counting the number of significant links between a given node and the rest of the graph. This has the advantage of removing the links with low connectivity. The threshold was set empirically to 0.2. In order to study the influence of the choice of threshold on the results, we also estimated the degrees using thresholds at 0.1 and 0.3. Overall the results are very similar (see Figure in supplementary material) and in the following, only results at a threshold of 0.2 will be reported.

Thresholding resulted in binary graphs (a link is 0 or 1), with each node corresponding to a channel, and a direction given by the delay. In other words, we assembled an asymmetric and directed adjacency matrix, based upon our measures of directed functional connectivity. Then, in each time window and for each channel, we counted the number of links with an h² value above the threshold, only for the outgoing links (OUT measure, corresponding to "out degrees" in graph theory), ingoing links (IN measure), and for all links independently of the directionality (TOT measure, with TOT=IN+OUT). High values of out-degrees OUT were considered to be indicative of the driving (i.e., source) regions [START_REF] Courtens | Graph Measures of Node Strength for Characterizing Preictal Synchrony in Partial Epilepsy[END_REF].(see also supplementary material for more details)

Graph comparison

For each link, the h 2 values were compared between PoS and PreS conditions. The test for a difference was performed across time windows (i.e., within subject) to compare two sets of functional connectivity measures for a given pairs of electrodes (one for PreS, one for PoS).

We also compared the degrees for each given region. For both comparisons, we used a Wilcoxon non-parametric paired test with Bonferroni correction for multiple comparisons.

Results

The clinical responses obtained in the three patients are summarized in Table 1. The seizure onset zone spared the insula in the three patients, but the insula was involved during the seizure spread. Patient 1 has already been reported in a previous study [START_REF] Picard | Induction of a sense of bliss by electrical stimulation of the anterior insula[END_REF]. Patients 2 and 3 also reported typical ecstatic sensations during their habitual seizures.

In all the three cases, the stimulated contacts were located in the antero-dorsal part of the insular cortex (two right side, one left side, Fig 1). The stimulations ranged from 1.6 to 2.1 mA (50 Hz, 1ms pulse width, 3-5 sec) and were not followed by any after-discharge. The patients were asked what they felt immediately after the stimulation ended. In two subjects a directed interview focusing on the ecstatic phenomenon was performed. In all three cases, it was not possible to precisely detect the timing of occurrence within or after the stimulation period but the ecstatic feeling lasted a few seconds. In the three patients, the other stimulations (particularly in the mesial temporal lobe) failed to trigger such phenomenon.

Figure 2A shows the functional connectivity graphs superposed on a 3D rendering of the cortex, with each bipolar channel representing a node of the graph. Links represents the strengths of h 2 values between pairs of nodes with a significative change (p <0.001190 for multiple comparison) between post stimulation (PoS) and pre stimulation (PreS) periods, during ecstatic symptoms induced by stimulation. These stimulations induced a significant increase of functional connectivity values between different regions, particularly between amygdala and hippocampus, dorsal anterior insula and prefrontal cortex, amygdala and orbitofrontal cortex, amygdala and dorsal anterior insula in P1; between dorsal anterior insula and orbitofrontal cortex, dorsal anterior insula and DLPFC, dorsal anterior insula and temporal neocortex, dorsal anterior insula and amygdala in P2; between dorsal anterior insula and amygdala, amygdala and DLPFC, amygdala and dorsolateral prefrontal cortex in P3.

Figure 2B presents the differences in mean total degree (number of significant links, independently of the direction of the links) between PreS and PoS periods. The following regions increased significantly their degree values: P1: amygdala, hippocampus, dorsal anterior insula, orbitofrontal cortex, dorsolateral prefrontal cortex; P2: dorsal anterior insula; P3: amygdala, dorsal anterior insula, premotor cortex, dorsolateral prefrontal cortex. Thus, variable changes in functional connectivity occurred after stimulation, but the most commonly implicated region in the three patients was the dorsal anterior insula. Out-degrees were estimated with a 0.2 threshold, in the PoS period. This measure is a mean of identifying leading structures in a network. The Fig 3A shows the out-degree values (colored spheres).

For P1, two regions presented the highest values, the dorsal anterior insula and the frontal lobe. In P2 and P3 the highest value was observed in the insular cortex. The out-degree values pooled from the three stimulations are shown in the Fig 3B showing that the insula was the structure with the largest changes in the three patients.

Discussion

We report here the induction of ecstatic auras by electrical stimulation of the dorsal anterior insula in 3 patients with refractory focal epilepsy. To our knowledge, these are the only descriptions of true ecstatic auras induced by brain electrical stimulation in the literature. In our 3 patients, no other stimulated areas than the dorsal anterior insula could induce such symptoms. We demonstrated that, at the time of the induction of the ecstatic symptoms, the insula was the most active region, playing a leading role among different areas forming an hyperactive network. Importantly, the analysis was performed in the few seconds following the stimulation, thus avoiding the bias related to the stimulation artifact.

The anterior insula has been recognized as a major component of the salience network, in association with the dorsal anterior cingulate cortex. The anterior insula activates in cases of behaviorally-relevant new external or internal stimuli [START_REF] Craig | How do you feel--now? The anterior insula and human awareness[END_REF][START_REF] Seth | Extending predictive processing to the body: emotion as interoceptive inference[END_REF]. The insula also integrates the interoceptive signals, i.e. stimuli coming from the inside of the body. It processes the physiological body state, contextualized by exteroceptive (environmental) signals. In the context of predictive coding in the brain, the insula continuously produces (top down) interoceptive predictions and generates prediction errors when the real signals arrive, allowing to update the next predictions. This process of interoception participates in the generation of emotions, feelings and probably the sense of self [START_REF] Craig | How do you feel--now? The anterior insula and human awareness[END_REF][START_REF] Seth | Extending predictive processing to the body: emotion as interoceptive inference[END_REF]. The neurocognitive hypothesis in ecstatic seizures is that an aberrant epileptic activity affecting a network which includes the anterior insula could prevent it from reaching the minimal level of differentiation and complexity necessary to encode predictions and/or prediction errors related to interoception and emotions. This would mimic a perfect match between interoceptive predictions and the incoming interoceptive signals, leading to a sense of certainty [START_REF] Picard | Predictions, perception, and a sense of self[END_REF][START_REF] Picard | State of belief, subjective certainty and bliss as a product of cortical dysfunction[END_REF]. Such a state was described as the "ultimate stable state", which Mumford suggested occurred when the topdown cortical signals would perfectly predict representations at lower cortical levels [START_REF] Mumford | On the computational architecture of the neocortex. II. The role of cortico-cortical loops[END_REF]. This would be the state of a perfect prediction of the world that the brain is trying to achieve, in order to avoid surprise and minimize energy expenditure.

There are two circumstances in which this sense of certainty and consequent bliss as experienced in ecstatic auras occurs. First, the "Aha!" moment or "eureka" moment (also known as insight) is an experience of sudden understanding of a previously incomprehensible problem or concept, accompanied by joy or satisfaction. A recent study showed a specific activation of the dorsal anterior insula at an eureka moment of bug detection in computer code [START_REF] Castelhano | The role of the insula in intuitive expert bug detection in computer code: an fMRI study[END_REF]. A previous study on insight solutions also showed an early bilateral insular activation [START_REF] Aziz-Zadeh | Aha!": The neural correlates of verbal insight solutions[END_REF]. Secondly, a peaceful and blissful state similar to the ecstatic state can be reached sometimes by meditation, in particular in the subjective experience of Samadhi during deep meditation or as the ultimate goal of yoga. Jamieson [START_REF] Jamieson | A unified theory of hypnosis and meditation states: the interoceptive predictive coding approach[END_REF] postulated that the non-reactivity to the contents of awareness in mindfulness practices leads to a heightened level of match between interoceptive predictions and the real felt state of the body [START_REF] Jamieson | A unified theory of hypnosis and meditation states: the interoceptive predictive coding approach[END_REF]. Mindfulness training was reported to induce an increased connection strength of the right insula in all its connections [START_REF] Sharp | Mindfulness training induces structural connectome changes in insula networks[END_REF]. A larger gyrification within the right antero-dorsal insula was also reported in meditators [START_REF] Luders | The unique brain anatomy of meditation practitioners: alterations in cortical gyrification[END_REF], as well as in increased grey matter volume in the left or right insula, or in the right fronto-insular cortex in a study on a yoga meditation practice [START_REF] Villemure | Neuroprotective effects of yoga practice: age-, experience-, and frequency-dependent plasticity[END_REF].

Our findings bring additional support in favor of a major role played by the dorsal anterior insula in the feeling of certainty, accompanied by a feeling of bliss and a sense of timelessness, as observed in ecstatic seizures. The fact that the stimulation of a certain part of the brain is able to induce the complex cognitive state observed in ecstatic auras is extremely interesting. The fact that we have shown an increased functional connectivity of the insula seems to exclude an inhibition of other regions by the abnormal activation of the insula, and favor the above-mentioned hypothesis of a hindrance to the generation of interoceptive prediction errors because of a supra-optimal level of activity inappropriate for this complex cognitive function. We must however recognize that these ecstatic phenomena have only been induced in patients with ecstatic epilepsy up to now, and that it is possible that epilepsy changes the excitability and wiring of the regions.

Legends of Table and Figures

Table 1. A: of the contacts inducing an ecstatic aura in the operculo-insular region in the 3 patients (P1, P2 and P3), in blue. The red dots correspond to other implanted electrodes.

Localisation was performed using in-house Gardel software [START_REF] Medina Villalon | EpiTools, A software suite for presurgical brain mapping in epilepsy: Intracerebral EEG[END_REF]. B: SEEG traces and an example of insular stimulation in patient 3. Note the absence of after discharges. Intracerebral electrodes are implanted under stereotactic conditions in Talairach's reference frame.

Electrodes are identified by letter (A, etc..) and the recordings leads are numbered from 1 to 15, low numbers corresponding to the deepest structures

  Abbreviations: M, male; F, female; N: normal; FCD: focal cortical dysplasia; OFC: orbitofrontal cortex; ES: epigastric sensation; R: right; L: left; TLE: temporal lobe epilepsy; FLE: frontal lobe epilepsy; ATL: anterior temporal lobectomy; Ant insula: anterior insula.

Figure 1 :

 1 Figure 1: Anatomical localization of the contacts in the dorsal anterior insula stimulated during ecstatic phenomenon.

Figure 2 .

 2 Figure 2. Intracerebral EEG functional connectivity changes in the three patients (P1, P2, P3)

Figure 3 :

 3 Figure 3: Estimation of the leading regions in the network. A: Spheres on each contact represent out-degree values according to the values indicated in the colour scale. B: Mean across time windows of total degree values pooled from the three stimulations (in the three patients). MTL: mesial temporal lobe, OFC: orbito-frontal cortex; TNcx: temporal neocortex. Other: corresponds to different electrodes in the different patients. Highest values are observed for dorsal anterior insula versus mesial temporal lobe (p=0.04), dorsal anterior insula versus temporal neocortex (p=0.04); orbitofrontal cortex versus temporal neocortex (p=0.005), dorsal anterior insula versus orbitofrontal cortex (p=0.005) and dorsal anterior insula versus other cortex (p=0.04) (Wilcoxon test with Bonferoni correction).

Acknowledgements

The authors thank Samuel Medina and Bruno Colombet for their help with Anywave and Gardel software.

Declaration of Interest:

Authors have no conflict of interest to disclose


Sovannarith San

MD Eleonore Ravis

MD Laetitia Tessonier

MD Mary Philip

MD Serge Cammilleri

MD Flora Lavagna

MD Giulia Norscini

MD Florent Arregle

Hélène Martel

MD Leopold Oliver

MD Olivier Torras

MD Sébastien Renard

MD Pierre Ambrosi

MD Laurence Camoin

PhD Anne Claire Casalta

MD Sandrine Hubert

MD Jean Paul Casalta

MD Frédérique Gouriet

MD Alberto Riberi

MD Jean-François Avierinos

MD Hubert Lepidi

PhD Frederic Collart

MD, Didier Raoult

PhD Michel Drancourt

Professor Gilbert Habib
email: gilbert.habib3@gmail.com

PhD Md

Prognostic Value of 18F-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography in Infective Endocarditis

Keywords: valve disease, endocarditis Condensed CT, 18 F-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography MSCT, multislice cardiac Tomography CRP, C-reactive protein IE, infective endocarditis IQR, interquartile range NVE, native valvular endocarditis PVE, prosthetic valve endocarditis TEE, transoesophageal echocardiographic TTE, transthoracic echocardiography

Introduction

Prognosis of infective endocarditis (IE) is poor with a still unacceptable reported inhospital (1-3) and long-term (4-6) mortality rate. Recent guidelines [START_REF] Habib | ESC Guidelines for the management of infective endocarditis: The Task Force for the Management of Infective Endocarditis of the European Society of Cardiology (ESC). Endorsed by: European Association for Cardio-Thoracic Surgery (EACTS), the European Association of Nuclear Medicine (EANM)[END_REF][START_REF] Baddour | Infective Endocarditis in Adults: Diagnosis, Antimicrobial Therapy, and Management of Complications: A Scientific Statement for Healthcare Professionals From the American Heart Association[END_REF] emphasize the benefits of a multidisciplinary approach led by the Endocarditis Team, the key role of an early surgery in the active phase of IE [START_REF] Botelho-Nevers | Dramatic reduction in infective endocarditisrelated mortality with a management-based approach[END_REF][START_REF] Thuny | The timing of surgery influences mortality and morbidity in adults with severe complicated infective endocarditis: a propensity analysis[END_REF], and the importance of an early prognostic assessment allowing an earlier management. Several prognostic factors have been identified such as advanced age, multiple comorbidities, heart failure, uncontrolled infection, embolic event, diabetes mellitus, renal insufficiency, staphylococcus-related IE, ischemic stroke or brain hemorrhage and echocardiographic findings [START_REF] Chu | Early predictors of in-hospital death in infective endocarditis[END_REF][START_REF] San Román | Prognostic stratification of patients with left-sided endocarditis determined at admission[END_REF][START_REF] Duval | Diabetes mellitus and infective endocarditis: the insulin factor in patient morbidity and mortality[END_REF]. However, the individual prediction of event is still difficult and additional prognostic markers may be useful to rapidly choose the best therapeutic option. 18 F-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography ( 18 F-FDG PET/CT) demonstrated to be able to improve the diagnosis of prosthetic valve endocarditis (PVE) [START_REF] Saby | Positron emission tomography/computed tomography for diagnosis of prosthetic valve endocarditis: increased valvular 18F-fluorodeoxyglucose uptake as a novel major criterion[END_REF][START_REF] Pizzi | Improving the Diagnosis of Infective Endocarditis in Prosthetic Valves and Intracardiac Devices With 18F-Fluordeoxyglucose Positron Emission Tomography/Computed Tomography Angiography: Initial Results at an Infective Endocarditis Referral Center[END_REF][START_REF] Rouzet | Respective performance of 18F-FDG PET and radiolabeled leukocyte scintigraphy for the diagnosis of prosthetic valve endocarditis[END_REF] increasing the sensitivity of the modified Duke criteria from 70% to 97 % [START_REF] Saby | Positron emission tomography/computed tomography for diagnosis of prosthetic valve endocarditis: increased valvular 18F-fluorodeoxyglucose uptake as a novel major criterion[END_REF].

Multi-imaging approach is nowadays essential in the diagnostic work out of IE [START_REF] Erba | Recommendations on nuclear and multimodality imaging in IE and CIED infections[END_REF][START_REF] Salaun | Diagnosis of Infective Endocarditis After TAVR: Value of a Multimodality Imaging Approach[END_REF] and has been integrated in the 2015 European Society of Cardiology criteria [START_REF] Habib | ESC Guidelines for the management of infective endocarditis: The Task Force for the Management of Infective Endocarditis of the European Society of Cardiology (ESC). Endorsed by: European Association for Cardio-Thoracic Surgery (EACTS), the European Association of Nuclear Medicine (EANM)[END_REF]. However, to date and to our best knowledge, no study focused on the prognostic value of 18 F-FDG PET/CT in IE.

Therefore, we aimed to assess the prognostic value of 18 F-FDG PET/CT in patients with PVE and NVE.

Methods

Population

From January 2011 to October 2017, all consecutive adults admitted in the Department of Cardiology in the teaching hospital La Timone (Marseille, France) with a definite diagnosis of left-sided IE were prospectively eligible to enter the study. Whether admitted for PVE or NVE, patients underwent a clinical 18F-FDG PET/CT which was realized as soon as possible after their admission. All patients had a follow up at 1, 3, 6 and 12 months after discharge including a physical examination, echocardiographic and biological evaluation. If the patient did not complete the follow up, the patient, his family or his cardiologist was reached by phone. Patient exclusion criteria were: incapacity to lie flat, pregnancy, need for urgent cardiac surgery, hemodynamic instability and blood glucose level >1.8 g/L at time of 18 F-FDG PET/CT, cardiac surgery during the 3 preceding months, right sided-IE or intra cardiac device infection and 18 F-FDG PET/CT performed more than 14 days after antibiotic initiation. Written informed consent was obtained from all patients using an approved protocol, which was validated by the institutional board.

Clinical and imaging data

Several data were collected during hospitalization and during follow up: age, sex, age and type of prosthetic valve, Charlson comorbidity index, history of diabetes, cancer, chronic renal insufficiency, high blood pressure, C-reactive protein level (CRP), causative pathogens, heart failure episode, embolic events (before treatment or under antibiotics), need for surgery and its indication, recurrence of IE, re-hospitalization, in hospital death and death during the 1 year follow up. Echocardiographic and multislice cardiac tomography (MSCT) data were also gathered. Transthoracic (TTE) and transesophageal echocardiographic (TEE) analyses were interpreted by experienced senior cardiologists. Echocardiographic data included the presence of vegetation, its length and mobility and the presence of paravalvular complications including abscesses, pseudo-aneurysm and fistula. Measurements of vegetation length were performed in various planes, and maximal length was used. Mobility was defined by vegetation with a fixed base but with a mobile free edge or pedunculated vegetation remaining within the same chamber or a vegetation prolapsing and crossing the coaptation plane of the leaflets during the cardiac cycle. An abscess was defined as a thickened area or mass with a heterogeneous echogenic or echo lucent appearance [START_REF] Habib | ESC Guidelines for the management of infective endocarditis: The Task Force for the Management of Infective Endocarditis of the European Society of Cardiology (ESC). Endorsed by: European Association for Cardio-Thoracic Surgery (EACTS), the European Association of Nuclear Medicine (EANM)[END_REF]. Recurrence included relapse due to a repeat episode of IE caused by the same microorganism and reinfection due to an infection by a different microorganism.

F-FDG PET/CT.

Patients fasted for 12 hours before 18 F-FDG PET/CT to limit physiological myocardial 18-FDG uptake. Patients received a low carbohydrate, high fat and protein meal before 18 F-FDG PET/CT (Discovery PET/CT 710, General Electric, Milwaukee, Wisconsin). Imaging started 60 min after 18-FDG injection (5mBq/kg) with a non-enhanced, low dose computer tomography scan (120kV, 80mA) with a whole-body PET acquisition in 3-dimensional (3D) mode.

Transverse PET slices were reconstructed into a 256x256 matrix using the OSEM (orderedsubset-expectation-maximisation) algorithm. The PET data were linked with the CT data. The data analysis (Xeleris, General Electric before 2013 and Advantage Workstation, General Electric after 2013) was based on visual interpretation. Assessment of 18-FDG uptake was performed by 2 experienced and blinded nuclear medicine physicians. Examination was considered positive when uptake was focal i.e less than 50% of the circumference's valve.

Otherwise examination was considered negative. Pathological uptake had to be confirmed in the uncorrected images. Abnormal valvular uptake was graded in 3 level scales: low intensity uptake when intensity in the valvular area was lower than the intensity in the liver, moderate intensity when it was equal or slightly superior and intense when uptake was far greater. If examination showed intense and homogenous myocardial uptake with poor images, patient was excluded from the study. Mean radiation dose for a whole-body 18 F-FDG PET/CT was 15mSv.

Definition of primary endpoint.

Primary endpoint was a composite of major adverse cardiac events, i.e. in-hospital and one-year death, recurrence of IE, acute cardiac failure, non-scheduled hospitalization for cardiovascular reason, and new embolic event under antibiotics.

Statistical analysis

Continuous variables were expressed as the median (interquartile range [IQR]) and compared using the t-test or Mann-Whitney U test when the former could not be performed.

Categorical data were expressed as numbers (percentages) and compared using the Chi-square or Fisher exact test when the former could not be performed. Predictors of 18 F-FDG PET/CT were analyzed by a logistic regression (logit model). Predictors of primary composite endpoint were analyzes using univariate and multivariate proportional hazard model (cumulative outcomes).

Hazards proportional assumption was evaluated by using the Schoenfed residual. Number of predictors to include in the multivariate analysis were determined using Akaike's information criterion. Smaller AIC values indicated a better model fit. Survival rates from admission to primary endpoint were summarized using Kaplan-Meier survival curve and log rank test were used to perform comparison between positive and negative 18

Results

Baseline characteristics.

Three hundred sixty-seven patients were hospitalized for IE and underwent a 18 The baseline characteristics are summarized in Table 1 for the overall population, the PVE and NVE groups. Echocardiographic paravalvular complications included abscesses (n=46/67, 69%), pseudo-aneurysm (n=20/67, 30%) and fistula (n=1/67, 1%). MSCT showed 4 abscesses, 5 pseudoaneurysms, 3 vegetations and one aortic cusp thickening. Theoretical indication for surgery was present in 127 (73%) patients and 54 % (n=93) finally underwent surgery.

Indications for surgery were heart failure (48%, n=61), prevention of embolism (30%, n= 38) and uncontrolled infection (22%, n=28).

F-FDG PET/CT findings.

Median time from first antibiotic to examination was 7 days (IQR: 4 to 8 days).

Characteristics of patients according to the 18 F-FDG PET/CT are shown in Table 2. 18 F-FDG PET/CT was positive in 58 % (n=100/173), more often in the PVE than in the NVE group (83% and 16% respectively). By univariate analysis, age, prosthetic valve, atrial fibrillation, CRP level and paravalvular complications were associated with a positive 18 F-FDG PET/CT, while male sex was associated with a negative 18 F-FDG PET/CT. By multivariable analysis, prosthetic valve (OR= 31 (10.8-93.1); p=0.0001), CRP (OR =1.1 (1.02-1,2); p=0.02) and presence of paravalvular complications (OR = 3.1 (1.2-8.2); p=0.02) were independent predictors of positive 18 F-FDG PET/CT.

In the PVE group (n=109), 19 false negative PET studies were observed. Median CRP level was lower in these patients (51.5 mg/L (24-80) as compared with the 90 PVE with positive PET/CT (89 mg/L (26-126)), p=0.04.

At time of admission, secondary septic locations were detected by 18 F-FDG PET/CT in 36% of patients (n=62/173), including splenic (n=24), spondylodiscitis or arthritis (n=24), pulmonary (n=20), vascular (n=5) or renal (n=2) uptakes. Portal of entry was identified in 16 patients by 18 F-FDG PET/CT, colic in 10 patients, dental in 3, cutaneous in 2 and on an orthopedic material in 1.

Incidence of primary endpoint and major cardiac events

At a mean follow-up of 225 days (IQR: 199 to 251 days), primary composite endpoint occurred in 94 (55%) patients, 63 (58%) in the PVE group and 31 (48%) in the native valve EI group (Table3). Median time from first day admission to composite endpoint was 45 days (IQR:18 to 115 days). Cumulative incidence of endpoint criteria at one-year follow-up was 53 % (95 % IC: 46% to 62 %) (Figure 2). Fourteen (8%) patients died during hospitalization from post-operative complication (n=4), infective complication (n=4) cardiogenic shock (n=4), and neurological complication (n=2). One-year mortality was 20% (n=35 patients), 25(23%) in the PVE group and 10 (16%) in the NVE group. Median time to death was 51 days (IQR: 30 to 142 days). Unplanned re-hospitalization during the year following admission occurred in 40 (23%) patients and was related to acute cardiac failure (n=20), septicemia (n=8), stroke (n=5), atrial fibrillation (n=3), atrioventricular block (n=2), vascular aneurysm (n=1) and acute coronary syndrome (n=1). Recurrence occurred in 15 patients (9%) including 5 relapses and 10 reinfections with another pathogen. Median time to recurrence was 104 days (IQR: 33 to 224 days). Symptomatic embolic events after treatment occurred in 22 (13%) patients at a median time of 14 days (IQR: 7 to 27 days). Location of embolism was cerebrovascular in 17 patients (77%), vascular in 3 (14%), involving superior mesenteric artery (2) and right coronary artery (1), hepatic (1) and renal [START_REF] Duval | Temporal trends in infective endocarditis in the context of prophylaxis guideline modifications: three successive population-based surveys[END_REF].

Prognostic value of 18 F-FDG PET/CT in the PVE group

Predictors of the primary endpoint are shown in Table 4. By univariate analysis, age ≥ 70 years, history of coronary artery disease, left ventricular ejection fraction ≤ 45 %, renal insufficiency at admission, staphylococcus aureus as causative pathogen, severe valve regurgitation, CRP level ≥ 100 mg/L, leukocytes count>10000/mm3, vegetation length > 10 mm, positive 18 F-FDG PET/CT and moderate to intense valvular 18 F-FDG PET/CT uptake were associated with increased rate of primary endpoint. The best model selected by AIC was a model with four predictors (CRP>100mg/L, severe valvular regurgitation, positive 18-FDG PET/CT and moderate to intense 18-FDG valvular uptake) (AIC=53.3). By multivariate analysis, CRP level ≥ 100mg/L, a positive 18 F-FDG PET/CT and a moderate to intense 18 F-FDG PET/CT valvular uptake remained associated with a higher rate of primary endpoint. Proportional hazards assumption was confirmed by the Schoenfed residual as the test was not statistically significant for each of the covariate (p>0.05) and the global test (p= 0.296). Central illustration (A,B)

shows the Kaplan-Meier survival curves and their comparisons for primary endpoint in case of positive 18 F-FDG PET/CT or in case of moderate to intense valvular uptake and cumulative incidence of primary-endpoint.

Positive 18 F-FDG PET/CT was not associated with each separate criterion of the primary composite endpoint (Table 5), except for new embolic events, which were more frequent in case of moderate to intense 18 F-FDG PET/CT valvular uptake in the PVE group (HR: 7.5; IC95%= 1.2 to 45.2; p=0.03) (Figure 3). New embolic events occurred in 16 patients with moderate to intense 18 F-FDG PET/CT uptake (1 in the NVE group and 15 in the PVE group) and 6 patients with negative or low 18 F-FDG PET/CT uptake (Figure 3A).

Prognostic value of 18 F-FDG PET/CT in the NVE group.

Predictors of primary endpoint are shown in Table 4. By univariate analysis, only diabetes mellitus, history of high blood pressure, vegetation length > 10 mm, and severe valve regurgitation were associated with increased rate of primary endpoint. 18 F-FDG PET/CT was not predictive of the primary composite endpoint whether considered positive or moderate to intense 18-FDG uptake. However, when displaying a moderate to intense uptake, 18 F-FDG PET/CT was associated with more frequent new embolic events in the NVE group (HR: 8.8; IC95%= 1.1-69.5; p=0.02) (Table 5, Figure 3B).

Discussion

To our best knowledge, this is the first study assessing the prognostic value of 18 F-FDG PET/CT in IE. In case of PVE, positive 18 F-FDG PET/CT was a predictor of bad outcome. In addition, positive 18 F-FDG PET/CT was also associated with a higher risk of new embolism both in the PVE and the NVE groups. Primary endpoint including all major cardiac events occurred in more than half of the patients (55%), confirming the need for systematic and regular patient's follow-up after discharge (4,5).

18 F-FDG PET/CT in infective endocarditis:

18 F-FDG PET/CT is now a recognized diagnostic method in suspected endocarditis, particularly in suspected PVE [START_REF] Saby | Positron emission tomography/computed tomography for diagnosis of prosthetic valve endocarditis: increased valvular 18F-fluorodeoxyglucose uptake as a novel major criterion[END_REF][START_REF] Pizzi | Improving the Diagnosis of Infective Endocarditis in Prosthetic Valves and Intracardiac Devices With 18F-Fluordeoxyglucose Positron Emission Tomography/Computed Tomography Angiography: Initial Results at an Infective Endocarditis Referral Center[END_REF][START_REF] Rouzet | Respective performance of 18F-FDG PET and radiolabeled leukocyte scintigraphy for the diagnosis of prosthetic valve endocarditis[END_REF][START_REF] Erba | Recommendations on nuclear and multimodality imaging in IE and CIED infections[END_REF][START_REF] Salaun | Diagnosis of Infective Endocarditis After TAVR: Value of a Multimodality Imaging Approach[END_REF][START_REF] Gomes | Diagnostic value of imaging in infective endocarditis: a systematic review[END_REF], both for the diagnosis of cardiac (major criterion) and peripheral (minor criterion) localization [START_REF] Habib | ESC Guidelines for the management of infective endocarditis: The Task Force for the Management of Infective Endocarditis of the European Society of Cardiology (ESC). Endorsed by: European Association for Cardio-Thoracic Surgery (EACTS), the European Association of Nuclear Medicine (EANM)[END_REF]. However, a meta-analysis showed that pooled sensitivity (when including NVE and PVE) of 18 F-FDG PET/CT for diagnosis of IE is only 76.8% and this relatively low accuracy was primarily due to the poor sensitivity for NVE [START_REF] Erba | Recommendations on nuclear and multimodality imaging in IE and CIED infections[END_REF]. Therefore, as recommended by current guidelines [START_REF] Habib | ESC Guidelines for the management of infective endocarditis: The Task Force for the Management of Infective Endocarditis of the European Society of Cardiology (ESC). Endorsed by: European Association for Cardio-Thoracic Surgery (EACTS), the European Association of Nuclear Medicine (EANM)[END_REF][START_REF] Baddour | Infective Endocarditis in Adults: Diagnosis, Antimicrobial Therapy, and Management of Complications: A Scientific Statement for Healthcare Professionals From the American Heart Association[END_REF], the use of 18 F-FDG PET/CT for IE diagnosis is only restricted to PVE. This was clearly confirmed by our study where sensitivity for PVE was much higher than for NVE. Several factors have been associated with a positive PET/CT in previous studies [START_REF] Mahmood | Meta-analysis of 18F-FDG PET/CT in the diagnosis of infective endocarditis[END_REF], including PVE, timing of the 18 F-FDG PET/CT study, and high inflammatory activity [START_REF] Swart | Improving the Diagnostic Performance of 18F-Fluorodeoxyglucose Positron-Emission Tomography/Computed Tomography in Prosthetic Heart Valve Endocarditis[END_REF]. In a recent study [START_REF] Swart | Improving the Diagnostic Performance of 18F-Fluorodeoxyglucose Positron-Emission Tomography/Computed Tomography in Prosthetic Heart Valve Endocarditis[END_REF], Swart et al. showed that low CRP level (<40mg/L) was predictive of false negative 18 F-FDG PET/CT. Similar results were observed in our series, in which median CRP levels were lower in 19 patients with PVE and false negative 18 F-FDG PET/CT, as compared with 90 patients with PVE and positive 18 F-FDG PET/CT. By multivariable analysis, only prosthetic valve, high CRP value and presence of paravalvular complications were independent factors of positive 18 F-FDG PET/CT. CRP level was an independent predictor of positive 18 F-FDG PET/CT both in the PVE group and in the overall population. Physician should be aware of the risk of false negative 18 F-FDG PET/CT in case of low systemic inflammation.

-18 F-FDG PET/CT predicts bad outcome in PVE:

Early and accurate prognostic assessment is a key issue in IE. In addition to conventional clinical, biological, and echocardiographic markers [START_REF] Chu | Early predictors of in-hospital death in infective endocarditis[END_REF][START_REF] San Román | Prognostic stratification of patients with left-sided endocarditis determined at admission[END_REF][START_REF] Thuny | Risk of embolism and death in infective endocarditis: prognostic value of echocardiography: a prospective multicenter study[END_REF], our study showed that 18 F-FDG PET/CT was able to provide independent information about the risk of occurrence of complications (heart failure, in-hospital and one-year death, recurrence, new embolism and rehospitalization) in patients with IE. We also observed a "dose-effect" relationship as a moderate to intense uptake was even more strongly associated with major cardiac event, as compared with negative or low intensity uptake. Interestingly, systemic inflammation measured by C-reactive protein was also an independent predictor of adverse cardiac events. Therefore, inflammation either systemic or focal has a key role in the prognosis of IE. High level of inflammation suggests poor outcomes and occurrence of major cardiac adverse event.

-18 F-FDG PET/CT predicts embolic events:

Embolic events are a frequent and life-threatening complication of IE related to the migration of cardiac vegetations. Although several factors have been associated with an increased risk of embolism [START_REF] Hubert | Prediction of symptomatic embolism in infective endocarditis: construction and validation of a risk calculator in a multicenter cohort[END_REF], its prediction is still difficult in the individual stratification. Our study showed that both in case of PVE or NVE, 18 F-FDG PET/CT was an independent predictor of new embolism. To date, the size of the vegetation is acknowledged to be the best predictor of new embolic event and our study shows that, independently of the vegetation size, the presence of a high level of local inflammation assessed by 18 F-FDG PET/CT is also predictive of new embolic event. Vegetation length and 18 F-FDG uptake seem to be two complementary predictors of new embolism. Therefore, in addition to echocardiographic findings, a positive 18 F-FDG PET/CT at time of admission might be considered as an additional indication for prophylactic surgery in patients with large vegetations. However, since our study is the first to have highlighted this finding, additional studies are warranted to demonstrate that the addition of a positive 18 F-FDG PET/CT to echocardiographic data at time of admission may help deciding the indication and timing of surgery in these patients.

Study limitations

Our study is limited by the relative low number of patients and events, particularly in NVE since the number of positive 18 F-FDG PET/CT was low in this group. Thus, the prognostic value of 18 F-FDG PET/CT has only been demonstrated in PVE with a composite endpoint but not with the in-hospital nor long-term mortality. Despite this limitation, we were able to demonstrate the value of 18 F-FDG PET/CT in predicting embolic events even if, given the low event count of new embolic event, statistical tests were performed without adjustment. Finally, our results cannot be applied in critically ill patients since they were unable to perform 18 F-FDG PET/CT, representing a selection bias.

Conclusion

This is the first study assessing the prognostic value of 18 F-FDG PET/CT. Our study showed that the nuclear imaging technique was predictive of major adverse cardiac event (in hospital death, one-year death, recurrence or IE, acute cardiac insufficiency, symptomatic embolism under antibiotics and non-scheduled rehospitalization for cardiovascular indication) in case of PVE. These results reinforce the utility of 18 F-FDG PET/CT in PVE, and justify its use in this population, not only for diagnostic purpose but also for prognostic assessment. 18 F-FDG PET/CT enables a prognostic stratification and should be used in clinical practice for optimal patient's management and therapeutic decision, particularly in PVE.

Perspectives

Competency in Patient Care and Procedural Skills: 18F-Fluorodeoxyglucose Positron Emission

Tomography /Computed Tomography (18F-FDG PET/CT) can be useful for diagnosis and estimation of prognosis in patients with infective endocarditis (IE).

Translational Outlook: Future studies should focus on defining the appropriate use of 18F-FDG PET/CT imaging in the evaluation of patients with IE.

Figure Legends

Central Illustration: Prosthetic Valve Endocarditis: Kaplan-Meier Curves. (A1) Kaplan-Meier curve at 1-year follow-up for the primary endpoint incidence in the PVE group comparing positive versus negative 18 F-FDG PET/CT. A2 shows cumulative incidence curve for the primary endpoint in case of positive 18 F-FDG PET/CT (62% (95% IC:53% to 73%)) or in case of negative 18 F-FDG PET/CT (38% (95% IC:27% to 47%)). (B1) shows Kaplan-Meier curve comparing negative or low 18 F-FDG uptake versus moderate to intense 18 F-FDG uptake. B2

shows cumulative incidence curve for the primary endpoint in case of moderate to intense 18 F-FDG uptake (64% (95% IC: 54% to 76%)) or of negative 18 F-FDG PET/CT or low 18 F-FDG uptake (36% (95% IC:26% to 46%)). Abbreviations. PVE: prosthetic valvular endocarditis. 18 F-FDG PET/CT: 18 Fluorodeoxyglucose Positron Emission/ Computerized Tomography. follow up in the overall population. The blue curve represents cumulative incidence of primary endpoint in the overall population and red curves its 95% confidence interval. Cumulative incidence was 54 % (95 % IC: 46% to 62 %) in the overall population. Figure 2B represents cumulative incidence of primary endpoint and its 95% confidence interval in the NVE group.

Cumulative incidence was 48 % (95 % IC: 38% to 62 %) in the NVE population. Figure 2C represents cumulative incidence in the PVE group. Cumulative incidence was 58 % (95 % IC: 48 % to 67 %) in the PVE population Abbreviations: NVE: native valvular endocarditis. PVE: Prosthetic valvular endocarditis. 18 F-FDG PET/CT: and in red, negative or low 18 F-FDG uptake versus intense uptake. Figure 3B shows survival curve without new embolism in the NVE group when comparing in blue negative versus positive 18 F-FDG PET/CT and in red negative or low 18 F-FDG uptake versus intense uptake. Abbreviations: NVE: native valvular endocarditis. PVE: Prosthetic valvular endocarditis. 18 F-FDG PET/CT: 18 Fluorodeoxyglucose Positron Emission/ Computerized Tomography.

  F-FDG PET/CT groups. Statistical analyses were separately conducted in the NVE and the PVE groups. Results with p values <0,05 were considered significant. Statistical analysis was conducted by Statview version 5.0 for Windows.

  F-FDG PET/CT study. We excluded one hundred twenty-two patients because IE involved cardiac devices, pulmonary or tricuspid valves. Flowchart of the study is shown in Figure 1. Median age was 67 years (interquartile range (IQR): 59-77 years old) and most of patients were men (70%).

Figure 1 :

 1 Figure 1: Flowchart of the study. Blue boxes indicate patients included in the final analysis.

Figure 2 :

 2 Figure 2: Primary Endpoint Incidence. Figure 2A shows B Kaplan-Meier curve at 1 year

Figure 3 :

 3 Figure 3: 18-FDG PET/CT as a Predictor of New Embolism. Figure 3A shows survival curve

TABLE 2 . Predictors of 18-FDG PET/CT results.

 2 Table 2 shows characteristics of patients according to 18-FDG PET/CT results. Abbreviations: BNP: brain natriuretic peptide; CRP: C-reactive protein; 18-FDG PET/CT: 18-fluorodeoxyglucose Positron Emission/ Computerized Tomography. PVE: prosthetic valve endocarditis

		Negative 18-FDG	Positive 18-FDG PET/CT	p-value
		PET/CT (n=73)	(n=100)
	Demographic data
	Age, year	65.8 (55.7-73)	71 (62-78)	0.04
	Male sex, n (%)	59(81)	61 (61)	0.007
	High blood pressure, n (%)	28 (38)	44 (44)	0.5
	Diabetes, n (%)	13 (18)	21 (21)	0.7
	Prosthetic valve	19 (26)	90 (90)	0.0001
	Atrial Fibrillation	18 (26)	38 (44)	0.03
	Clinical data
	Fever at admission, n	21 (44)	36 (36)	0.5
	Leukocytes count, mm3	8.9(6.8-11)	9.6(7.6-13)	0.2
	Creatinine level serum, µmol/L	84 (72-117)	95 (76-129)	0.2
	BNP, ng/l	237 (129-462)	299(133-549)	0.4
	CRP, mg/L	56 (23-98)	75(29-129)	0.05
	Positive blood culture	53 (73)	75 (75)	0.7
	Vegetation >10mm	24 (33)	37(37)	0.5
	Paravalvular complication	14 (19)	51 (51)	0.0001
	Time from first antibiotic to	7 (4-8)	6(4.5-8.0)	0.57
	PET/CT, days
	Primary outcome endpoint, n (%)	26 (54)	30(30)	0.6

TABLE 3 INCIDENCE OF PRIMARY ENDPOINT AND MAJOR CARDIAC EVENTS

 3

		All (n=173)	PVE group (n=109)	NVE group (n=64)
	Primary endpoint, n (%)	94 (55)	63 (58)	31 (48)
	In-hospital death, n (%)	14 (8)	9 (8)	5 (8)
	1-year death, n (%)	35 (20)	25 (23)	10 (16)
	Recurrence, n (%)	15 (9)	8 (7)	7 (11)
	New embolic event, n (%)	22 (13)	16 (15)	6 (9)
	Acute cardiac failure, n (%)	66 (38)	33 (30)	33 (51)
	Re-hospitalization, n (%)	40 (20)	27(20)	13 (20)

Incidence of primary endpoint and major cardiac events in the overall population, the prosthetic valve endocarditis (PVE) group and native valve endocarditis (NVE) group.

		HR (IC95%)	p value HR (IC95%)	p	HR (IC95%)	p value HR (IC95%)	p value
					value
	In-hospital death	0.49 (0.5 -2.6)	0.36	0.43 (0.1 -1.8)	0.25	2.78 (0.44-17.5)	0.25	1.53 (0.16-16.1) 0.69
	One year death	1.43 (0.41-6.0)	0.5	1.4 (0.51-3.97)	0.5	1.35 (0.26-8.06)	0.68	1,77 (0,64-4.93) 0.27
	Recurrence	1.24 (0.14-10.98)	0.84	3.1 (0.37-26.28) 0.28	2.45 (0.4-14.85)	0.67	3.0 (0.35-26)	0.3
	New embolic	3.1 (0.37-26)	0.82	7.5 (1.24-45.2)	0.03	3.13 (0.5-20)	0.21	8.8 (1.1-69.5)	0.02
	event
	Acute cardiac	1.97 (0.6 -0.45)	0.25	1.0 (0.44-2.57)	0.89	1.87 (0.56-6.24)	0.31	1.3 (0.54-3.13)	0.55
	failure
	Re hospitalization 3.27 (0.7-15.2)	0.11	3 (0.94-9.47)	0.05	2.5 (0.53-11.9)	0.24	3.57 (1.1-11.3)	0.03

Major cardiac events according to the 18-FDG PET/CT findings in the PVE and NVE groups. Abbreviations. HR: hazard ratio. 18-FDG PET/CT: 18- fluorodeoxyglucose Positron Emission/ Computerized Tomography; NVE: native valvular endocarditis.