Mathieu Chappuy
email: mathieu.chappuy@chu-lyon.fr

Benoit Trojak

Philippe Nubukpo

Jérôme Bachellier

Patrick Bendimerad

Georges Brousse

Benjamin Rolland

Prolonged-release buprenorphine formulations: Perspectives for clinical practice

Keywords: Buprenorphine, Prolonged-release, Opioid dependence, Opiates, Substitution ADR: adverse drug reaction OAT: opioid agonist treatment OUD: opioid use disorder PRB: prolonged-release buprenorphine

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Opioids constitute a category of substances that act on receptors of the same name. Opioids are widely used in medicine, in particular because of their powerful analgesic effects. Depending on the dose used and the mode of action, opioids also induce and calm though intense euphoria, and they can thereby be used with a recreative purpose, or in self-medication of different types of symptoms. In both cases, this corresponds to what is called 'misuse' [START_REF] Strang | Opioid use disorder[END_REF]. In practice, all opioids can be associated with misuse, but medical opioids are commonly distinguished from illicit opioids, for example heroin, or fentanyl derivatives such as ocfentanil or carfentanil. By way of illustration, it is estimated that a few more than 500,000 people in France have ever used heroin [START_REF]French Monitoring Centre for Drugs and Drug Addiction -Drugs[END_REF].

The main risk of opioid misuse is the occurrence of overdose. The effects of sedation and respiratory depression are very strong with opioids, and the risk of death is serious in case of overdose [START_REF] Strang | Opioid use disorder[END_REF]. For example, each year in France, between 400 and 500 people die from opioid overdose [START_REF]French Monitoring Centre for Drugs and Drug Addiction -Evolution of the number of overdose deaths since 1985[END_REF].

When opioids are injected, their use is also associated with a great risk of viral or bacterial infection, in particular regarding the human immunodeficiency virus (HIV), and the hepatitis B and C viruses (HBV and HCV) [START_REF] Strang | Opioid use disorder[END_REF]. Furthermore, opioid misuse may lead to opioid use disorder (OUD), which correspond to developing criteria for addiction [START_REF] Strang | Opioid use disorder[END_REF]. Regular use of opioids rapidly leads to neuroadaptation processes, which induce the occurrence of tolerance and withdrawal symptoms, thus impeding the ability of the subject to stop or reduce opioid use [START_REF] Strang | Opioid use disorder[END_REF]. Moreover, progressive processes of behavioral habituation settle down, strengthening the need for using opioids and the occurrence of craving and loss of control over the substance. Such processes are met both with illicit and prescription opioids [START_REF] Dematteis | Recommendations for buprenorphine and methadone therapy in opioid use disorder: a European consensus[END_REF][START_REF] Rolland | Misuse and dependence on prescription opioids: Prevention, identification and treatment[END_REF].

OUD is associated with moderate to severe psychosocial consequences, which thus requires a specialized treatment [START_REF] Strang | Opioid use disorder[END_REF]. This treatment is based both on an individualized psychosocial support and on the use of an opioid agonist treatment (OAT) [START_REF] Strang | Opioid use disorder[END_REF]. OATs are maintenance drugs that have no recreative effects in therapeutic use, while they allow to restrain withdrawal symptoms and craving, and that reduce the psychosocial consequences of OUD. For these reasons, they are now considered as the gold standard pharmacotherapy of OUD [START_REF] Dematteis | Recommendations for buprenorphine and methadone therapy in opioid use disorder: a European consensus[END_REF][START_REF] Volkow | Prevention and treatment of opioid misuse and addiction: a review[END_REF].

In France, two OATs are labeled for OUD: 1) methadone, and 2) high dose buprenorphine.

Methadone was the first drug to be used in this indication, as soon as the 1960s [START_REF] Dole | A medical treatment for diacetylmorphine (heroin) addiction. A clinical trial with methadone hydrochloride[END_REF]. Methadone is a full-agonist of the mu opioid receptor, and is has a very long pharmacological action, with an average half-life of 25 hours [START_REF] Strang | Opioid use disorder[END_REF]. Methadone is more effective than placebo for supporting the cessation of heroin use and the associated aftermaths of heroin use, but in practice, methadone can also expose patients to an increased risk of overdose, and patients treated with methadone should thus be strictly supervised [START_REF] Kleber | Methadone maintenance 4 decades later: thousands of lives saved but still controversial[END_REF]. However, the overdose risk in patients under medical supervision is probably less important than in untreated heroin users, and the risk/benefit ratio of methadone in OUD is frequently considered as favorable by the experts of the field [START_REF] Dematteis | Recommendations for buprenorphine and methadone therapy in opioid use disorder: a European consensus[END_REF][START_REF] Sordo | Mortality risk during and after opioid substitution treatment: systematic review and meta-analysis of cohort studies[END_REF]. Buprenorphine is the alternative OAT that has been marketed in France. Until recently, the galenic forms of buprenorphine have consisted of sublingual tablets, which allow to avoid the hepatic first-pass effects. By contrast with methadone, buprenorphine is a partial agonist of the mu receptor.

Consequently, the risk of overdose, and thus the risk of death related to overdose, is very limited with buprenorphine [START_REF] Kimber | Mortality risk of opioid substitution therapy with methadone versus buprenorphine: a retrospective cohort study[END_REF]. However, much more than methadone, buprenorphine can be diverted by injection or intranasal use. Such buprenorphine misuses can lead to medical consequences, such as viral infections or distal microvascular infringements [START_REF] Lofwall | A review of buprenorphine diversion and misuse: the current evidence base and experiences from around the world[END_REF].

In France, the use of OAT has started in the mid1990s. A clear public health preference has been made on access to treatment, in particular with regard to buprenorphine [START_REF] Auriacombe | French field experience with buprenorphine[END_REF]. Initiating buprenorphine treatment has been permitted to any physician, including office-based general practitioners. Moreover, buprenorphine can be prescribed for 28 days, whereas methadone syrup can be prescribed only for 14 days. For these reasons, buprenorphine has been the most prescribed OAT in France, while the rate of treatment coverage among opioid users has been very high, as it has been estimated that between 70 and 80% of them receive more or less frequent prescriptions of OAT, essentially of buprenorphine [START_REF] Fatseas | Why buprenorphine is so successful in treating opiate addiction in France[END_REF].

Despite the relative success of the 'French way' of managing the public health strategy of OUD treatment, many issues remain with regard to this population. Many patients are not sufficiently treated as they do not receive adequate doses or are not correctly educated on how to use buprenorphine.

Buprenorphine tablets remain too frequently swallowed by patients, which dramatically restrain the bioavailability of the drug [START_REF] Elkader | Buprenorphine: clinical pharmacokinetics in the treatment of opioid dependence[END_REF]. Meanwhile, sublingual tablet use may require up to ten minutes to be dissolved under the tongue, which can be tedious and may cause underdosing, and thus foster the injection or intranasal misuse of buprenorphine [START_REF] Maremmani | Buprenorphine dosing choices in specific populations: review of expert opinion[END_REF], even if a lyophilizate of buprenorphine has recently been commercialized in France, which substantially reduces the sublingual dissolve time, and enhances the bioavailability of the drug [START_REF] Lyseng-Williamson | Buprenorphine oral lyophilisate (Espranor®) in the substitution treatment of opioid dependence: a profile of its use[END_REF].

Furthermore, treatment dropouts remain frequent in this population. Release from prison or hospitalization are at risk situations, in which treatment follow-up and continued access to OAT prescriptions can be difficult to ensure. Such situations expose patients to resuming illicit opioid use, or purchasing OATs on the black market, with increased risks of overdose, in particular with methadone.

A last major concern in France is that buprenorphine misuse remains frequent, as it might concern 20% of patients at the international level [START_REF] Lofwall | A review of buprenorphine diversion and misuse: the current evidence base and experiences from around the world[END_REF], and up to 50% of them in French specialized addiction centers [START_REF]French Monitoring Centre for Drugs and Drug Addiction -Opioid Agonist Treatment Dashboard 2019 Update[END_REF].

In this context, new galenic forms of 'prolonged-release' buprenorphine are becoming available in Western countries. These new treatments consist either of subcutaneous depot forms, or in implants.

They allow for a very long treatment coverage, i.e., between one week to six months. In France, at least one of these specialties should be available very soon. This article aims to describe the main characteristics of these new medicines, the main evidence regarding efficacy and safety features, and the different expectancies and possible concerns of opioid users and professionals regarding the use of prolonged-release buprenorphine (PRB).

Characteristics of the different formulations of prolonged-release buprenorphine

PRB can be divided in two main formulations: 1) subcutaneous implant, and 2) subcutaneous injection depot. Three medical specialties are currently available at the internal level: one implant (Probuphine ® / Sixmo ® ) and two depot formulations (Sublocade ® and Brixadi ® / Buvidal ® ) [Table 1].

The main features of the three different specialties can be found in Table 2, and will be detailed below.

Sixmo ® / Probuphine ® : the six-month implant by Titan Pharmaceuticals ®

Characteristics of the product

This product is the first implant of buprenorphine. It consists of four white / light yellow implants (Ethylene vinyl acetate copolymer) that are 26.5 mm long and have a 2.4 mm diameter. Each of them delivers 74.2 mg buprenorphine over a six-month period for a total amount of 296.8 mg, even if the packaging of the drug only specifies the amount of dose per implant. The implants are to be implanted in the internal part of the higher part of the patient's arm, beneath the shoulder, using a local anesthesia.

The implants must be surgically removed after six months. This surgical procedures of placing and removing the implant has to be undertaken by a trained physician. This specialty is already commercialized in the USA, and a European labeling has recently been granted (brand name: Sixmo ® ) [START_REF]Sixmo ® : summary of product characteristics[END_REF]. Sixmo ® will be indicated for "clinically stabilized" (i.e., at least 30 days without heroin use) adults, in whom buprenorphine oral daily dose does not exceed 8 mg. The treatment duration is limited to two successive implants, that is, 12 months. After that, is planned to switch back to oral form. Moreover, in some circumstances (e.g., stress, craving, withdrawal symptoms), an adjunctive treatment of sublingual buprenorphine can be prescribed. In this respect, the summary of product characteristics (SmPCs) specify as follows: "Although some patients may require occasional supplemental dosing with buprenorphine, patients should not be provided with prescriptions for sublingual buprenorphine-containing products for as needed use. Instead, patients who feel the need for supplemental dosing should be seen and evaluated promptly" [START_REF]Sixmo ® : summary of product characteristics[END_REF]. In the clinical trials, 22.5% patients received additional doses of sublingual buprenorphine, and 21.9% of them required a 5 th implant [START_REF] Rosenthal | Buprenorphine implants for treatment of opioid dependence: randomized comparison to placebo and sublingual buprenorphine/naloxone[END_REF].

Efficacy / safety data Three efficacy studies were conducted among 627 subjects [START_REF] Rosenthal | Buprenorphine implants for treatment of opioid dependence: randomized comparison to placebo and sublingual buprenorphine/naloxone[END_REF][START_REF] Ling | Buprenorphine implants for treatment of opioid dependence: a randomized controlled trial[END_REF][START_REF] Rosenthal | Effect of buprenorphine implants on illicit opioid use among abstinent adults with opioid dependence treated with sublingual buprenorphine: a randomized clinical trial[END_REF]. The first two studies [START_REF] Rosenthal | Buprenorphine implants for treatment of opioid dependence: randomized comparison to placebo and sublingual buprenorphine/naloxone[END_REF][START_REF] Ling | Buprenorphine implants for treatment of opioid dependence: a randomized controlled trial[END_REF] were double blind randomized placebo-controlled trials. Efficacy was defined as a negative urine screen at the end of the study period, i.e., 24 weeks for 287 patients (31 % for Sixmo ® versus 13 % placebo versus 33 % for sublingual buprenorphine) and 16 weeks for 163 patients (40 % versus 28 % placebo).

Participants should not be treated with buprenorphine or another OAT before the inclusion in the study.

The third trial [START_REF] Rosenthal | Effect of buprenorphine implants on illicit opioid use among abstinent adults with opioid dependence treated with sublingual buprenorphine: a randomized clinical trial[END_REF] was also a double-blind double-dummy randomized trial, but the control group consisted of subjects treated with sublingual buprenorphine. This trial enrolled 177 subjects who were already treated with sublingual buprenorphine before inclusion, with a maximum daily dose of 8 mg, which certainly explain why this is the maximum dose required as the indication of the final approval.

After 6 months of treatment, approximately 96% were negative for heroin in the group receiving the active implant, versus 88% in the group receiving active sublingual buprenorphine, which confirmed the non-inferiority of Sixmo ® , compared to the sublingual galenic form.

No concerning safety issue was reported in these three trials. The most frequent adverse events were those of buprenorphine, whatever be the galenic formulation. Specific adverse events related to the implant form were reported, but they were not serious. They essentially consisted of local pain, itching, bleeding, or reddening at the implantation site. Such minor symptoms affected approximately one quarter of participants, which is not a negligible figure.

Sublocade ® : Indivior's prolonged-release depot formulation (unknown European trade name)

Characteristics of the product

This specialty is planned for 2021 in the EU. By contrast with Sixmo ® , Sublocade ® is not an implant but a depot formulation. Buprenorphine is encapsulated in a biodegradable polymeric gel (50:50 poly(DL-lactide-co-glycolide) polymer and a biocompatible solvent, N-methyl-2-pyrrolidone) named Atrigel ® . Sublocade ® has to be subcutaneously injected. This procedure does not require a specific previous training. Moreover, the gel spontaneously dissolves and no removal procedure is required.

Sublocade ® depot has a therapeutic action of one month. Two dosages exist: 1) 100 mg (in 0.5 mL solution); and 2) 300 mg (in a 1.5 mL solution). Two successive injections must be separated by at least 26 days of delay. Sublocade ® has to be stored in a refrigerator (at between +2 and +8°C), as its stability does not exceed 7 days at room temperature. Kits are displayed as pre-filled syringes which a secure needle has only to be fixed on. Injection is performed in the abdominal area, remotely from the umbilicus.

In the US, Sublocade ® is approved for subjects with OUD that have been "stabilized" for at least 7 days with a sublingual form of buprenorphine, using a dose ranging from 8 to 24 mg per day [START_REF]Sublocade ® : summary of product characteristics[END_REF]. The dosing scheme consists of delivering a 300 mg monthly formulation during the first two months, followed by a maintenance period based on a monthly 100 mg dosing. However, the American SmPCs provide for the possibility of maintaining a monthly 300 mg regimens in patients that would not wish to receive 100 mg, while they specify that the controlled trials have not demonstrated the superiority of using 300 mg monthly versus using 100 mg (see below).

In France, the drug circuit and the dispensing procedure (i.e., take-home, or restricted to treatment settings) is still unknown, as the French drug agency has not stated on this point.

Efficacy / safety data

The efficacy study that was conducted in view of obtaining the approval was a 6-month long, 489subject, randomized controlled trial [START_REF] Haight | Efficacy and safety of a monthly buprenorphine depot injection for opioid use disorder: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial[END_REF]. Participants were separated into three groups that received: 1) a monthly 300 mg Sublocade ® injection during 6 months; 2) 300 mg per month during two months, followed by 100 mg monthly during the four following months; and 3) placebo during six months, respectively. The main assessment criterion was reporting no opioid use and displaying a negative urine screen at the end of the study period (41.3% with 300 mg monthly were abstinent versus 42.7% with 300 mg then 100 mg versus 5.0% with placebo). Important differences were found, between the two Sublocade ® and the placebo group, but there was no difference between the two schemes of Sublocade ® use. Though this study design was previously approved by the Food and Drugs Administration (FDA), and certainly discussed with Indivior ® before the conductance of the trial, it is very unlikely that this kind of design will be accepted by the European Medicines Agency. In Europe, it is generally required to compare a new drug, or a new galenic form, with the standards of care, that is, here, with sublingual buprenorphine. Nevertheless, clinical studies comparing Sublocade ® with actives comparators (buprenorphine without naloxone which is the most used form in our country and methadone) are announced (EudraCT Number: 2018-004460-63), as well as studies exploring the efficacy initiating Sublocade ® in subjects that were not previously treated with sublingual buprenorphine (NCT03993392).

Moreover, a study conducted among 39 patients explored the PK and pharmacodynamics features of Sublocade ® [START_REF] Nasser | Sublocade then published in sustained-release buprenorphine (RBP-6000) blocks the effects of opioid challenge with hydromorphone in subjects with opioid use disorder[END_REF]. A peak concentration is reached 24 hours after administration. Blood concentrations of buprenorphine were of 5.0 ng per mL and was slightly higher after the second injection (6.6 ng per mL). Sublocade ® could block the effects of 6 and 18 mg of hydromorphone (i.m), which is a mu-receptor agonist. As a partial mu-receptor agonist exhibiting a very high affinity for this receptor, buprenorphine is able to block the effects of many full agonists. Sublocade ® have allowed for a significant reduction in the reinforcing effects of hydromorphone. This test using hydromorphone is an efficacy criterion requested by the Food and Drug Administration. Based on these results, it shows that the buprenorphine contained in Sublocade ® is effective for targeting the mu-opioid receptors of patients.

Overall, the tolerability of Sublocade ® was acceptable. Local adverse drug reactions affected 16.5% of participants versus 9.0% of those with placebo [START_REF]Sublocade ® : summary of product characteristics[END_REF], and consisted of local pain, itching, or erythema. Other adverse drug reactions comprised the common effects of buprenorphine, e.g., constipation, nausea, vomiting, headache, or sedation. After commercialization, and according to a recent financial report issued by Indivior [START_REF] Indivior | Financial results[END_REF], 10,630 people had received at least one Sublocade ® injection in mid2019. 74% of them had a second administration, while 56% had received three injections, 39% had five injections, and 21% ten injections. This may point out a substantial rate of dropouts, but, meanwhile, the commercialization is also recent, and a limited number of subjects were able to receive an important number of injections at this stage.

A multi-center, open-label, long term clinical study was conducted with the aim to assess the effectiveness and safety up to 12 months of Sublocade ® [START_REF] Ling | Effects of monthly buprenorphine extended-release injections on patient-centered outcomes: A long-term study[END_REF]. Different measures were collected during the study (EQ-5D-5L, SF-36v2, treatment effectiveness assessment (TEA), addiction severity index-lite (ASI-Lite), employment/insurance status questionnaire and medication satisfaction questionnaire (MSQ). 402 patients were included and 50% have been lost to follow-up. Regarding quality of life, EQ-5D-L remained stable, but SF-36v2 increased with a significant mental improvement. Improvement was noted for all treatment effectiveness assessment domain score. For the addiction severity indexlite, significant improvements were seen for all problem areas except for alcohol use from baseline to end of study. The proportion of participants employed increased 7% from base-line to end of study (44.2% vs 51.2%) whereas the proportion with health insurance was stable from baseline to end of study. At end of study, 88.8% of participants stated they were satisfied with treatment.

Buvidal ® : Camurus ® ' weekly or monthly prolonged-release depot formulation

Characteristics of the product

Buvidal ® is the first prolonged release buprenorphine depot formulation that has received a European labeling. The encapsulation of buprenorphine is made into a lipid-based liquid named FluidCrystal ® (soybean phosphatidylcholine and glycerol dioleate). Like for Sublocade ® , the depot form spontaneously dissolves in the body, and does not require a removal procedure. In France, this specialty should be commercialized in 2020. Buvidal ® is displayed either with weekly forms, with four different dosages of 8, 16, 24, or 32 mg, or monthly forms of 64, 96, or 128 mg. Buvidal ® galenic aspect consists of a secure pre-charged syringe, which only requires the piston to be screened on. The injected volume is between 0.16 and 0.64 mL for the weekly form, and 0.18 and 0.36 mL for the monthly form.

Injection can be delivered in the buttock, the leg, the arm, or the abdomen.

The correspondence between Buvidal ® dosing and the daily sublingual doses of buprenorphine is not linear a requires using an equivalence table that is displayed in the SmPCs (Table 3) [START_REF]Buvidal ® : summary of product characteristics[END_REF]. This equivalence table is based on "classical" sublingual forms of buprenorphine with limited bioavailability, while more recent galenics (lyophilisates form) may have a higher bioavailability [28], which should be considered when using the correspondence table. Moreover, no equivalence table between Buvidal ® and Sublocade ® dosages is currently available, and there is thus no indication yet, on how to proceed the switch from one form to the other.

Buvidal ® has been approved for treating OUD in adults as well as in adolescents aged 16 years or more. Setting up an adjunctive psychosocial support is required in the SmPCs. Contraindications are similar to those of the sublingual form.

Similarly to Sublocade ® , the dispensing process of Buvidal ® is unknown at this stage, as the regulatory procedure is still ongoing.

Efficacy / safety data

The efficacy of Buvidal ® has been explored one multicenter double-blind double-dummy randomized clinical trial in which Buvidal ® was compared with sublingual buprenorphine / naloxone during 24 weeks among 428 subjects [START_REF] Lofwall | Weekly and monthly subcutaneous buprenorphine depot formulations vs daily sublingual buprenorphine with naloxone for treatment of opioid use disorder: a randomized clinical trial[END_REF]. First group was treated with daily sublingual placebo and weekly (first 12 weeks; phase 1, up to 32 mg/week) and monthly (last 12 weeks; phase 2, up to 160 mg/month) Buvidal ® versus daily active sublingual buprenorphine (up to 24 mg/day) with naloxone (24 weeks) with matched weekly and monthly placebo injections for the second group. The main assessment criterion was the use of illicit opioid at the end the study, based on self-report and urine screen.

Participants should not be treated with buprenorphine during the 60 days prior to inclusion. This study found that Buvidal ® was not inferior to sublingual formulation for supporting the cessation of non-therapeutic opioid use. During the 24-week trial, 35.1% subjects in the Buvidal ® active group, versus 28.4% in the sublingual buprenorphine / naloxone active group, displayed continuous negative urine screens. When combined with self-reports, these proportions fell at 17.4% and 14.4%, respectively.

A long-term safety study was conducted [START_REF] Frost | Long-term safety of a weekly and monthly subcutaneous buprenorphine depot (CAM2038) in the treatment of adult outpatients with opioid use disorder[END_REF]. It was a 48-week-long multicenter trial conducted among 227 subjects. Participants received individualized doses, and could be treated using either the weekly or monthly formulation. 37 participants started buprenorphine with the depot form, whereas 190 switched from sublingual to depot form. One third of the sample received only weekly depot injections, whereas one other third received only monthly injections, and the other third received weekly and then subsequently monthly injection formulations. Approximately 83% of the participants ended the study. When taken together, urine screens and self-reports found that 63% of the participants initiated with Buvidal ® , and 82.8% of the participants who had switched from sublingual buprenorphine to Buvidal ® , maintained cessation of non-therapeutic use of opioids.

A study conducted among 47 patients explored the pharmacokinetic and pharmacodynamics features of Buvidal ® weekly forms (24 mg and 32 mg) [START_REF] Walsh | Effect of buprenorphine weekly depot (CAM2038) and hydromorphone blockade in individuals with opioid use disorder: a randomized clinical trial[END_REF]. A peak concentration is reached after 24 hours, with a half-life of 4 to 5 days. Four hours after administration, blood concentrations of buprenorphine were of 1.25 ng per mL or more, while it is stated that between 2 and 3 ng per mL are required to block the majority of the mu receptors. On a clinical ground, all withdrawal symptoms disappeared within the first 24 hours. Moreover, Buvidal ® could block the effects of 6 and 18 mg of hydromorphone, which is a mu-receptor agonist. As a partial mu-receptor agonist exhibiting a very high affinity for this receptor, buprenorphine is able to block the effects of many full agonists. With these results, it shows that the buprenorphine contained in Buvidal ® is effective.

Regarding the safety aspects, the 24-week-long trial [START_REF] Lofwall | Weekly and monthly subcutaneous buprenorphine depot formulations vs daily sublingual buprenorphine with naloxone for treatment of opioid use disorder: a randomized clinical trial[END_REF] found that 31.3% reported adverse drug reactions, both groups combined. The tolerability features related to the injections were essentially light to moderate pain (8.4%), itching (6.1%), or erythema (5.6%), at the injection site. Other adverse drug reactions consisted of headache (7.7%), constipation (7.5%), or nausea (7.5%). Five cases of overdose were reported, all of them in the active sublingual arm. The observational study [START_REF] Frost | Long-term safety of a weekly and monthly subcutaneous buprenorphine depot (CAM2038) in the treatment of adult outpatients with opioid use disorder[END_REF] found that 63% of the participants reported at least one adverse event, among which 26.4% of them could be related with Buvidal ® . These were local pain (15.4%), swelling (11.9%), and erythema (9.3%) at the site of injection. Other general adverse drug reactions were headache (7.9%), nausea (7.0%), and vomiting (5.3%). Surprisingly, patients switched from sublingual to depot form reported two-fold more adverse drug reactions than those directly treated with Buvidal ® . Five participants (2.2%) were withdrawn from the study because of a safety issue. The third study [START_REF] Walsh | Effect of buprenorphine weekly depot (CAM2038) and hydromorphone blockade in individuals with opioid use disorder: a randomized clinical trial[END_REF] found relatively similar figures.

Prolonged-release buprenorphine: expectancies and concerns of users and professionals

Opioid users

International surveys

So far, only four surveys have investigated the expectancies and preferences regarding prolongedrelease buprenorphine among people who use opioid drugs.

In the UK, a qualitative survey was conducted in 2018 among 44 opioid users who had received a prolonged-release buprenorphine treatment (implant or depot form) [START_REF] Neale | Implants and depot injections for treating opioid dependence: qualitative study of people whouse or have used heroin[END_REF]. Surveyed participants expressed some apprehensions regarding the possible coercive risk of these new galenic formulations that are difficult to stop overnight, compared to sublingual form. Such remarks are in line with some frequent concerns expressed by opioid users, who commonly worry about the risk of social and /or medical control upon their behaviors of drug use [START_REF] Vorspan | What place for prolonged-release buprenorphine depot-formulation Buvidal® in the treatment arsenal of opioid dependence? Insights from the French experience on buprenorphine[END_REF]. In this context, any treatment scheme that is deemed as too rigid or abstinence-focused can be perceived as a constraint. Nevertheless, the people interviewed in this survey also raised a relative interest for prolonged-release buprenorphine formulations. In particular, they pointed out that these new galenic forms could break the daily ritual of taking buprenorphine or another OAT, which could help some subjects to feel more easily away from an identity of "addicted", and thereby more swiftly fit into a recovery trajectory.

A second British qualitative survey has explored the perceptions on prolonged-release buprenorphine of 36 subjects receiving daily oral OAT [START_REF] Neale | Prolonged-release opioid agonist therapy: qualitative study exploring patients' views of 1-week, 1-month, and 6-month buprenorphine formulations[END_REF]. In this sample, the perceptions on such new formulations were quite favorable. Participants deemed that these new formulations will be useful to put distance with other opioid users and medical settings, and thus to recover a more "normal" life.

Conversely, they thought that these new specialties would not attract people who are not willing to completely stop opioid use, and /or would not want to completely cut themselves from a users' environment. Ethical concerns were also raised, in particular the same risk of coercion addressed in the first survey.

In Australia, a recently-published quantitative survey conducted among 402 users of opioids found that the overall acceptation of prolonged-release buprenorphine was correct [START_REF] Larance | Perceptions of extendedrelease buprenorphine injections for opioid use disorder among people who regularly use opioids in Australia[END_REF]. More than two third of the participants considered that these new forms constituted an interesting therapeutic option for them. Compared to uninterested subjects, those who reported an interest in these long-acting forms were younger, more frequently females, and had a lower level of education. They were more recent opioid users. A clear preference was reported for monthly formulations, compared to weekly regimens.

The issue of how far to go to access buprenorphine was also a major factor in adherence to PRB.

Last, preliminary results of the French study 'AMBRE' were recently published [START_REF] Bachellier | A-t-on besoin de traitements de substitution «long acting» en France ? SWAPS-Chemex[END_REF]. This was a multicenter quantitative survey conducted among 366 French opioid users treated with an OAT, between February and July 2019. 55% of the participants (probably those with split deliveries) reported that the main constraint with their oral OAT was the need to frequently have to meet a prescriber and/or a delivery pharmacist. If weekly injections will not change this feeling, for these patients, the monthly version might give them more flexibility. The need to take an OAT every day was also an issue for 40% of them. One third of participants reported more or less frequent problems with the intake of current forms, e.g., intimacy, professional constraints, or travelling issues. Approximately a half of the interviewed sample was ready to try a prolonged-release buprenorphine formulation. The main cause of interest for these subjects was the weekly or monthly aspect of the treatment (86.7% of the interested subjects), and the expected reduction in craving (81.2%) or withdrawal symptoms (80.3%). They also considered these new treatment forms as more discreet than sublingual tablets (73.3%).

Opinions on opioid users' online forums

Opioid users already debate on prolonged-release formulations of buprenorphine on community online forums (for example, in France, [START_REF]Buprénorphine injectable[END_REF][START_REF]Subutex : une injection par mois par l'infirmière[END_REF][START_REF]Buprénorphine en implant sous-cutané[END_REF]). For some of them, injection OATs should not be long-acting, but more immediate-release buprenorphine, for people who inject themselves their sublingual buprenorphine treatment. Some opinions expressed on forums are in line with what was found in the qualitative surveys, i.e., that the subjects with OUD that will be the most interested in prolongedrelease buprenorphine would those that wish to completely stop recreative opioid use and aim for a complete social reinsertion. Some interesting concerns were also raised about the risk of feeling bad at the end of the injection action, i.e., that blood levels of buprenorphine would be low and induce withdrawal symptoms and / or craving.

Professionals involved in the treatment of opioid use disorder

Overall, most of the specialists deem that the treatment coverage of OUD is insufficient, and that new galenic forms are thus required [START_REF] Strang | Opioid use disorder[END_REF][START_REF] Dematteis | Recommendations for buprenorphine and methadone therapy in opioid use disorder: a European consensus[END_REF][START_REF] Ling | Depot buprenorphine injection in the management of opioid use disorder: from development to implementation[END_REF]. A recent expert opinion article, which only pertained to Buvidal ® , but could easily apply to other formulations, listed several types of specific publics for prolonged-release buprenorphine (Table 4) [START_REF] Vorspan | What place for prolonged-release buprenorphine depot-formulation Buvidal® in the treatment arsenal of opioid dependence? Insights from the French experience on buprenorphine[END_REF]. However, they also stated that these new formulations should always be proposed, and never imposed to patients with OUD, with respect to the apprehensions addressed above.

The reception that general practitioners already have, or will have in France, with regard to prolonged-release buprenorphine formulations remain largely unknown. In France, this question is important, because general practitioners are by far the most frequent prescribers of OATs, as it has been explained in the Introduction section. However, in France as abroad, an insufficient rate of general practitioners accepts to be involved in the prescribing of OATs [START_REF] Rolland | Misuse and dependence on prescription opioids: Prevention, identification and treatment[END_REF][START_REF] Vorspan | What place for prolonged-release buprenorphine depot-formulation Buvidal® in the treatment arsenal of opioid dependence? Insights from the French experience on buprenorphine[END_REF]. They might fear to prescribe a treatment such as sublingual buprenorphine, which can lead to traffic or diversion. In such case, they will maybe feel more at ease with prolonged-release buprenorphine formulations, which theoretically have no risk of misuse. By contrast, they may also feel uncomfortable with injecting constraints associated to these specialties. Surveys among general practitioners would be helpful to better address their knowledge and representations on these new treatments. In France, the drug circuit of the long-acting forms has not been specified at this stage. Two options are supposed: 1) a wide outpatient-based access to the medicine, which is administered by a health professional (i.e., a doctor or a nurse), with the risk that the product may be diverted after delivery in pharmacies; or 2) a restricted access to addiction settings, which reduces the likelihood of misuse, but which will also greatly restrict the access to this galenic forms. In this second option, the financial coverage of these drugs will impact the budgets of many addiction units, which will further restrict the access to these new types of drugs. A final possible option would that training community pharmacists would perform the injection, as they already do for influenza vaccination. This option would allow a wider access while avoiding misuse, but it is unsure that pharmacists are ready for this.

If the French High Authory for Health (HAS) has still not decided which place these new galenics will have in the country, the NICE (British agency) has stated as follows [START_REF]Opioid dependence: buprenorphine prolongedrelease injection (Buvidal)[END_REF]: "Buprenorphine prolonged-release injection may be an option where there is a risk of diversion of opioid substitution medicines or concerns about the safety of medicines stored at home. It may also be an option for people who have difficulties adhering to daily supervised opioid substitution medication, such as for people who are working or in education.

Buprenorphine prolonged-release injection may have a place in treating opioid dependence in people in custodial settings, where the risk of diversion and time needed for supervised consumption currently leads to challenges in supplying supervised medicines safely.

However, the higher drug acquisition cost of buprenorphine prolonged-release injection compared with other treatments for opioid dependence will need to be taken into account". In the United Kingdom, a monthly supply of buprenorphine prolonged-release injection costs £239.70 (excluding VAT about 275€) either approximatively the price of buprenorphine high dosage whereas methadone is much cheaper (£15 to £30). In France, we do not know the price expected by pharmaceutical companies.

Conclusions and perspectives

Prolonged-release buprenorphine will soon be available in France. Buvidal ® has been approved and should arrive on the market in 2020. Sublocade ® should follow quite quickly. These two specialties have many similarities, in particular in terms of tolerability and easiness of use, even if Buvidal ® has the advantage to be stored at ambient temperature. These two products have also some differences (see Table 2). Only Buvidal ® has a weekly formulation, though in practice, the monthly injection seems to be more expected by opioid users. Buvidal ® has also a wider range of dosages, which can be swifter for clinical practice. Concerning Sixmo ® , though the possibility of a six-month-acting product can be very appealing for some subjects, it can also be repelling for some others. But the authorization of only two treatments before returning to the sublingual form is very odd and difficult operationalize in current practice. Moreover, the constraints related to placing and removing the implants may refrain many physicians and many patients from using this product. All put together, these products may attract a very heterogeneous public of patients, from those living in places with difficult access to delivery pharmacies or prescribers from those bothered by the need to take a drug daily. Some unknown questions will also need to be addressed with these new formulations. For example, the practical management of an acute and severe pain is likely to occasionally lead to pitfalls or issues. The potential of misuse and diversion of these products is theoretically very low, but this remains to be confirmed such cases will have to be declared to the regional addictovigilance center (For France: [START_REF] Ceip | [END_REF] in order to monitor this potential for post-market abuse. On a more general ground, postmarketing data of phase IV and pharmacovigilance follow-up will be necessary to guarantee the safe use of these products on the long run. Furthermore, the dose correspondence between the three specialties would need to be assessed, even if, for other long-acting treatment, e.g., antipsychotics, such correspondence were never established without too many practical issues.

Disclosure of interest

MC: ATHS Congress (2017): invitation by Indivior ® as a participant (inscription fees, travelling and housing costs). ATHS Congress (2019): invitation by Ethypharm ® as a participant. JB: conflicts of interests with Camurus ® , Indivior ® , Bouchara-Recordati ® , HAC ® and Shire ® GB: conflicts of interests with Indivior ® , Bouchara-Recordati ® , Lundbeck ® , Janssen-Cilag ® , Sanofi ® and AstraZeneca ® . BR: conflicts of interests with Ethypharm ® , Camurus ® , Indivior ® , Bouchara-Recordati ® , Lundbeck ® , Otsuka ® , Grunenthal ® , HAC ® and Shire ® .

  BT: Encephale Congress (2020) and ATHS Congress (2019): invitation by Camurus ® as a participant (inscription fees, travelling and housing costs). ECNP Congress (2019) invitation by Jansen-Cilag ® as a participant (inscription fees, travelling and housing costs). PB: conflicts of interests with Indivior ® , Ethypharm ® , ATHS Congress (2019): invitation by Abbvie ® as a participant (inscription fees, travelling and housing costs). Albatros (2019) and Encephale Congress (2020): invitation by Camurus ® as a participant, Clinical survey (Ambre) participation for Camurus ® . PN: did not report competing interest with this manuscript.

Abbreviations

 4. Situations and publics for which prolonged-release buprenorphine could be particularly fitted (expert opinion [START_REF] Vorspan | What place for prolonged-release buprenorphine depot-formulation Buvidal® in the treatment arsenal of opioid dependence? Insights from the French experience on buprenorphine[END_REF])

Situations Examples

People concerned with avoiding daily OAT intake (practical aspects and stigma)

-living in the parental home, in a reintegration home, in incarceration -frequent travelers, especially abroad -reinserted subject wishing to limit their contacts with the drug consumer environment and the healthcare environment (pharmacy and doctor), and no longer take daily medication People having difficulty ensuring daily buprenorphine taking and motivated to do it.

-precarious social situation, entourage consumer of recreational opioids Take care relay with risk of interruption -out of prison, from reintegration home, from psychiatric hospitalization -a move out People with diversion their OAT or selling a part of them and wishing to stop


P Dumas

S Duguay

J Borrel

Atom probe tomography quantification of carbon in silicon

Atom Probe Tomography (APT) was used to quantify carbon in implanted silicon at two various electric fields (~ 15 and 20 V/nm). Using equal proportions of implanted 12 C and 13 C, the numerous molecular ions that were observed were identified and their contribution to the carbon content statistically derived. Much more accurate carbon quantification was obtained in the lowest electric field analysis by comparing APT with Secondary Ion Mass Spectroscopy profiles. This was assigned to a lower amount of molecular ion dissociations. Furthermore, the number of self-interstitials trapped per carbon atom in clusters was derived. This value of interest for the microelectronics industry regarding dopant diffusion and implantation induced defects was estimated close to one, in agreement with the expected stoichiometry of the SiC phase present in the phase diagram. However, this was obtained only when using low electric field conditions.

Introduction

Quantification of carbon in materials using Atom Probe Tomography (APT) is a tricky issue. Large underestimation in the carbon content is indeed observed [START_REF] Thuvander | Quantitative atom probe analysis of carbides[END_REF][START_REF] Estivill | Quantitative analysis of Si/SiGeC superlattices using atom probe tomography[END_REF][START_REF] Peng | On the detection of multiple events in atom probe tomography[END_REF].

One well known reason of carbon biases arises from the occurrence of multiple events and related detection biases. This occurs when several ions strike the detector nearly simultaneously and close to each other [START_REF] Peng | On the detection of multiple events in atom probe tomography[END_REF]. When this happens, a part of ions may be lost due to the so-called electronic "dead time" and "dead zone" existing between each hit [START_REF] Peng | On the detection of multiple events in atom probe tomography[END_REF][START_REF] Jagutzki | Multiple hit readout of a microchannel plate detector with a three-layer delay-line anode[END_REF][START_REF] Meisenkothen | Effects of detector dead-time on quantitative analyses involving boron and multi-hit detection events in atom probe tomography[END_REF]. This phenomenon is very likely when analyzing carbon because the high electric field needed to induce its evaporation promotes correlated departures of carbon ions at the tip surface [START_REF] Thuvander | Quantitative atom probe analysis of carbides[END_REF][START_REF] Peng | On the detection of multiple events in atom probe tomography[END_REF]. Another source of carbon multi-events is the dissociation of molecular ions during their flight from the tip to the detector [START_REF] Yao | On the multiplicity of field evaporation events in atom probe: A new dimension to the analysis of mass spectra[END_REF][START_REF] Peng | Unraveling the Metastability of C n 2+(n= 2-4) Clusters[END_REF].

An additional complication comes from the detection of carbon ions sharing the same massto-charge ratio, like (C2) 2+ and C + . When this happens, the proportion of each overlapped ion is needed to make a correct quantification. In some cases, it is possible to derive theses proportions from isotopes abundance [START_REF] Thuvander | Quantitative atom probe analysis of carbides[END_REF][START_REF] Martinavičius | Mechanism of Si3N4 precipitation in nitrided Fe-Si alloys: A novel example of particle-stimulated-nucleation[END_REF][START_REF] Lefebvre | Atom probe tomography: put theory into practice[END_REF]. However, in the field of ion implantation, only one isotope is selected though the mass analyzer. As 12 C accounts for 98.93 % of the natural isotopic repartition, 12 C + ions are usually selected to minimize the process time. As a result, mass-to-charge ratio overlaps cannot be solved using the isotope proportion method. For materials featuring the natural repartition of carbon (metallic alloys and other semiconductors), peak overlaps of minor species can still be solved at the expense of a higher level of uncertainty [START_REF] London | Quantifying uncertainty from mass-peak overlaps in atom probe microscopy[END_REF]. This uncertainty is lower if both 12 C and 13 C isotopes are present in similar proportions.

In this study, the issue faced using isotope abundance to solve carbon overlaps in low concentrated silicon will be overcome by implanting 12 C + and 13 C + in equal proportion. A statistical approach will be used to identify the molecular ions and quantify their contribution to the implanted concentration at two electric fields. The ambition is to clarify the contribution of various bias sources in overall biases. Although this study has been carried in carbon implanted silicon, results and conclusions may be transposed to carbon quantification in general.

Another aim of this work is to study carbon clustering. In the microelectronics industry, carbon is co-implanted in silicon to suppress transient enhanced diffusion of interstitially mediated dopants (boron and phosphorus) and the formation of extended defects [START_REF] Dumas | 3D atomic-scale investigation of carbon segregation in phosphorus co-implanted silicon[END_REF][START_REF] Cristiano | Interstitial trapping efficiency of c+ implanted into preamorphised silicon-control of EOR defects[END_REF][START_REF] Cacciato | Dislocation formation and B transient diffusion in C coimplanted Si[END_REF]. Carbon is indeed known to consume self-interstitials, notably through the formation of clusters. The number of self-interstitials trapped per carbon atom in clusters, or in other words the atomic fraction of carbon in clusters, will be here measured using APT.

Materials and method

12 C + and 13 C + ion implantations were performed at room temperature in a (100) oriented 300 mm silicon wafer. Doses, energies and tilts used were 1 ± 0.01 x 10 15 ions cm -2 , 30 keV and 3° respectively in the objective to get both 12 C and 13 C profiles very close to each other and centered around 100 nm in depth with a maximum carbon content of 0.2 at. %. Because increasing the number of implantation cycles is thought to lead to a better superimposition between 12 C and 13 C profiles, the total dose D (2 ± 0.02 x 10 15 ions cm -2 ) was implanted in 4 cycles, each cycle composed of one 12 C + sub-implant at D/8 followed by one 13 C + sub-implant at D/8. The wafer was then annealed at an average temperature of 750 °C for 5 h. Details of the annealing sequence applied are found in [START_REF] Dumas | 3D atomic-scale investigation of carbon segregation in phosphorus co-implanted silicon[END_REF]. In addition to restoring silicon crystallinity, this sequence is known to induce carbon clustering. Afterwards, carbon concentration profile was obtained from a CAMECA IMS dynamic Secondary Ion Mass Spectroscopy (SIMS) using a cesium beam at 4 keV, a beam current of 100 nA , and a sputtering rate of 0.41 nm/s. SIMS quantification was assessed from ion implantation doses.

A 1 cm 2 sample was then cut-off from the annealed wafer for APT tip preparation. Using a G4 CXe Dualbeam plasma focused ion beam, a small volume of annealed silicon was lifted-out and attached to a tungsten needle. Two sharp tips of around 30 nm diameter were then obtained from this volume using ion beam milling (see Fig. 1). Such small diameters are needed to generate the high electric field (~20 V/nm) that is required in APT analyses. These latter were conducted using a Laser-Assisted-Wide-Angle Tomographic Atom Probe (CAMECA-LAWATAP). The laser was operated at a 343 nm wavelength under ultra-vacuum conditions (~10 -10 Torr) at a 100 kHz repetition rate and was focused on a 0.1 mm spot diameter. The tip temperature was maintained at 80 K. After preparation and APT analyses, Xe is found to be below detection limits of APT.

Results and discussion

APT analyses were conducted using two laser powers (10 and 80 nJ/pulse) to elucidate the influence of the evaporation field on carbon quantification. The two mass spectra plotted in Fig. 2 represent the normalized number of detected ions as a function of the mass-to-charge ratio, at a given laser power. Kingham curves and Si + /Si 2+ ratio were used to assess electric fields at the tip surface [START_REF] Kingham | The post-ionization of field evaporated ions: A theoretical explanation of multiple charge states[END_REF]. They were found to be close to 15 and 20 V/nm and will be denoted hereinafter respectively as the lowest (and highest) electric field analysis.

Si + Si 2+ C + C 2+

Normalized counts

Mass-to-charge ratio (Da) Fig . 2. Mass spectra related to the analysis of C implanted silicon samples a) highest electric field analysis (10.160 million atoms and Si + /Si 2+ = 0.02) b) lowest electric field analysis (6.089 million atoms and Si + /Si 2+ = 1.7). Counts are normalized to the total number of atoms in each analyzed volume.

First, aside the expected presence of monoatomic C + and C 2+ ions from 6 to 13 Da (Fig. 2 a) b)), numerous other peaks were detected from 20 to 75 Da in the lowest electric field analysis (Fig. 2 b)). The latter are likely to be related to molecular ions evaporated from carbon-enriched clusters that nucleate due to carbon supersaturation. The carbon solubility limit in silicon, around 2 ppb at 750°C [START_REF] Goesele | Diffusion engineering by carbon in silicon[END_REF], is indeed very low compared to the carbon amount. The same range of peaks is not visible in Fig. 2 a) as it is hidden by the tails of 28 Si + and 28 Si 2+ peaks. These so-called thermal tails are related to 28 Si + and 28 Si 2+ ions evaporated after laser pulses during the cooling of the tip [START_REF] Kelly | Laser pulsing of field evaporation in atom probe tomography[END_REF][START_REF] Vella | Laser-Assisted Field Evaporation[END_REF]. This phenomenon is enhanced when using higher electric fields [START_REF] Hatzoglou | Preferential evaporation in atom probe tomography: an analytical approach[END_REF] (i.e. lower laser powers at a given evaporation rate) as highlighted in Fig. 2 a).

A cluster identification was applied to the lowest electric field using the iso-position method [START_REF] Marquis | On the use of density-based algorithms for the analysis of solute clustering in atom probe tomography data[END_REF]. The purpose of this identification was not quantitative but to build a mass spectrum less noisy than that in Fig. 2 b), to make the peaks related to molecular ions more distinct and their identification easier. The objective is to exclude from the analysis silicon atoms that are not present in peaks related to molecular ions (from 20 to 75 Da). To optimize the signal/noise ratio in the cluster identification process, only the well-defined peaks related to 12 C +/2+ and 13 C +/2+ were considered. The concentration threshold was taken to 0.05% and the minimal number of atoms per cluster to 5. Once again, these parameters do not necessarily account for the real size or the volume fraction of the clusters but were chosen to build a less noisy mass spectrum (Fig. 3). For identification purpose, detected ions were classified by families labelled as (SinCp) q+ where n and p respectively represent the number of silicon and carbon atoms, and q is the ion charge number (Fig. 3). For instance, the C + family comprises 12 C + and 13 C + ions. Applying the same cluster identification to data related to the highest electric field analysis allowed to identify molecular ions which were previously hidden by the Si + and Si 2+ tails. However, only 4 molecular families involving carbon were detected : (C(H20)2) 2+ , (C2) + , (SiC2) + and (SiC) + against 13 in the lowest electric field analysis.

For a given (SinCp) q+ family, we define its multiplicity as the number of different ways to form (SinCp) q+ ions from silicon and carbon isotopes. For example, for n=0, the multiplicity of the (C2) + family is three as it is composed of ( 12 C 12 C) + , ( 13 C 13 C) + and ( 12 C 13 C) + ions. A general formula is derived as follows. Let us first consider a family composed of k identical elements having z isotopes. Its multiplicity, also called multiset coefficient in combinatorics [START_REF] Stanley | Enumerative Combinatorics[END_REF], is equal to which denotes the number of combinations of k out of z + k -1. For a given (SinCp) q+ family, its multiplicity labelled γ(n,p) is then the product of γ(n,0) and γ(0,p): The multiplicity of pure molecular ions composed of p carbon ions is simply equal to p + 1.

, = 3 + -1 2 + -1 = + 1 + 2 +
Equation ( 1) is helpful to avoid missing contributions in peak identification, especially for large mixed families. It is worth mentioning that the observed number of peaks relative to a family is always lower or equal to its multiplicity. This is due to overlaps between peaks of the considered family that have the same mass-to-charge ratio. For instance, the number of peaks relative to the (SiC) + family is equal to 4 (at 40, 41, 42 and 43 Da) whereas γ(1,1) = 6 from equation [START_REF] Thuvander | Quantitative atom probe analysis of carbides[END_REF]. There are indeed 6 different types of ions belonging to the (SiC) + family but four of them share the same mass-to-charge ratio at 41 and 42 Da (see When there is an overlap, each contribution at the considered peak can be statistically derived from isotopic proportions. For this purpose, we define f(n28,n29,n30,p12) as the proportion of counts of a given type of ion composed of n28 28 Si, n29 29 Si, n30 30 Si , p12 12 C and (p -p12) 13 C relative to its family (SinCp) q+ . This latter is derived from isotopic natural abundances and combinations:

, , , = 1 2 -
! " #$ 2
Where z28, z29 and z30 are the natural proportions of silicon isotopes taken to 92.22%, 4.69%, and 3.09%, respectively. Proportions of carbon isotopes were obtained by comparing 12 C and 13 C SIMS profiles (Fig. 5). Centered around 100 nm, profiles have nearly gaussian shapes which are typical of ion implantation. Additional peaks around 60 and 75 nm are likely to reveal carbon segregations to extended defects, like those detected in [START_REF] Dumas | 3D atomic-scale investigation of carbon segregation in phosphorus co-implanted silicon[END_REF]. Furthermore, an 12 C accumulation is observed from the surface to 25 nm. This is attributed to an atmospheric contamination. 13 C contamination being around 100 times smaller than that of 12 C from isotopic natural proportions, it is screened by 13 C implanted concentration. Despite the difference at the surface, 12 C and 13 C SIMS profiles are practically superimposed all over the implantation axis. The fact that 12 C and 13 C are in equal proportions is expressed in equation ( 2) by the % term. A numerical application is given in Table 1. It shows that the repartition of the (SiC) + family is mainly governed by ions containing 28 Si, the most abundant silicon isotope. Note that in this example, proportions were simply obtained by dividing natural proportions of silicon isotopes by 2, as 12 C and 13 C are in equal proportions.

Examples of overlaps resolution between ions from different families are given in the following. ( 13 C 13 C) + and ( 28 Si 12 C 12 C) 2+ ions were detected at 26 Da. ( 13 C 13 C) + counts are half ( 12 C 13 C) + counts in agreement with equation (2). Because there is no overlap at 25 Da, ( 13 C 13 C) + counts are also half 25 Da counts. ( 28 Si 12 C 12 C) 2+ counts were then obtained from the difference between 26 Da and ( 13 C 13 C) + counts. For each peak, background counts were accounted locally by defining another element of the same mass window in the close vicinity of the considered peak [START_REF] Lefebvre | Atom probe tomography: put theory into practice[END_REF]. Background counts were then subtracted to obtain the true number of counts relative to each ion. Due to 13 C low natural abundance (1.07%), no peak is usually detected at 12.5 Da leaving the overlap between (C2) 2+ and C + ions unsolved. However, because 12 C and 13 C are here in equal proportions, ( 12 C 13 C) 2+ ions accounts for 50% of the overall counts of (C2) 2+ family. As there is no peak at 12.5 Da in both mass spectrum, we conclude that (C2) 2+ ions are absent or below the detection limit in our experimental conditions.

(C3) + ions have also been detected at 36, 37, 38 and 39 Da (Fig. 2 b)). Peak at 36 Da is the most pronounced among (C3) + peaks whereas the proportion of ( 12 C 12 C 12 C) + ions should be equal to ( 13 C 13 C 13 C) + relative to (C3) + . This highlights the presence of another type of ions at 36 Da that we have interpreted as ((H2O)2) + . The relative importance of (C3) + family in the overall carbon content can still be deduced, for example from the peak at 38 Da.

Complete carbon quantification was done through decorrelations of various overlapped ions. Families respective contributions to the carbon content were sorted in mass-to-charge ratio increasing order and plotted in a histogram (Fig. 4). The highest (respectively lowest) electric field analysis contributions are concentrated to the left (respectively right) hand side of the histogram. This trend indicates that the contribution of molecular ions is well lower in the highest electric field analysis (45% against 90%). We believe that this difference is attributed to the promotion of molecular ion dissociations at higher electric fields [START_REF] Peng | On the detection of multiple events in atom probe tomography[END_REF][START_REF] Peng | Unraveling the Metastability of C n 2+(n= 2-4) Clusters[END_REF]. This would explain why molecular families at high mass-to-charge ratios (> 40 Da) were not detected in the highest electric field analysis. Note that the above assumption is difficult to prove in our experimental conditions. First, no dissociation tracks were observed in the correlation diagrams of both analyses because of the very low carbon content (~0.1%). Second, reporting the proportion of total events that are multi events does not seem relevant as the number of identified family related to carbon differs between the two analyses (Fig. 4). Indeed, it is very likely that ions from different families do not have the same probability to induce multiple events. Still, assuming that the following dissociation reaction (C2) 2+ C + + C + reported in Peng et. al [START_REF] Peng | On the detection of multiple events in atom probe tomography[END_REF] occurred, a higher proportion of multi C+ events should reflect a higher probability of (C2) 2+ dissociation. 8,15% of C + events that are multi-events were reported in the lowest electric field analysis against 17.64% in the highest electric field analysis. This result support the assumption that more molecular ions dissociations related to carbon occurred in the highest electric field analysis. Contribution are sorted in mass-to-charge ratio increasing order.

APT carbon concentration profiles versus implantation depth were plotted in Fig. 5. SIMS profile has been calibrated from the implanted carbon dose (2 ± 0.02 x 10 15 ions cm-2 ). APT concentration profiles were built using sampling slices 1 nm thick and derived from the atomic volume of silicon (0.02 nm 3 ). The measured SIMS dose integrated over 175 nm is equal to 1.93 x 10 15 ions cm-2 whereas that derived from APT experiments are 1.47 x 10 15 ions cm-2 and 8.51 x 10 14 ions cm-2 for the lowest and highest electric field analysis respectively. Much more accurate quantification is obtained using low electric field conditions. As assumed previously, it is thought that dissociation of unstable molecular ions is promoted at higher electric fields. As a result, because multi-events occur after dissociation, more ions composed of silicon and/or carbon may have been lost in the detection process in the highest electric field analysis [START_REF] Peng | On the detection of multiple events in atom probe tomography[END_REF][START_REF] Peng | Unraveling the Metastability of C n 2+(n= 2-4) Clusters[END_REF]. The impact of the latter on the silicon content is thought to be negligible because only about 0.4% of silicon ions were detected in the form of molecular ions in both analyses. In contrast, this is not the case for carbon as 45% of its content was detected in the form of molecular ions (Fig. 4). Another composition-based cluster identification [START_REF] Marquis | On the use of density-based algorithms for the analysis of solute clustering in atom probe tomography data[END_REF] was applied to both analyses in the objective to reveal carbon enriched-clusters below the implantation peak and carbon segregations at structural defects [START_REF] Dumas | 3D atomic-scale investigation of carbon segregation in phosphorus co-implanted silicon[END_REF][START_REF] Cristiano | Interstitial trapping efficiency of c+ implanted into preamorphised silicon-control of EOR defects[END_REF]. Defects and related carbon segregations are not expected in the first 50 nm as no redistribution in the carbon SIMS profiles is observed (Fig. 5). From these considerations, the composition threshold of the cluster composition was set at 5% so as to exclude carbon ions originating from the first 50 nm. The minimum number of atoms per cluster Nmin was then determined by observing the reconstructed images for various increasing values of Nmin. Setting Nmin at 7 solute atoms was found reasonable so as to avoid tiny fortuitous clusters.

The atom map revealing carbon ions after cluster identification for the lowest electric field analysis is shown in Fig. 6. First, carbon segregations from 50 to 100 nm reveal structural defects that nucleate during recrystallization of a specific type of ion implantation induced amorphous layer [START_REF] Jones | A systematic analysis of defects in ion-implanted silicon[END_REF][START_REF] Dumas | 3D atomic-scale investigation of carbon segregation in phosphorus co-implanted silicon[END_REF]. The first 50 nm that were excluded from cluster identification corresponds in fact to recrystallized silicon in which carbon lie in solid solution [START_REF] Dumas | 3D atomic-scale investigation of carbon segregation in phosphorus co-implanted silicon[END_REF][START_REF] Strane | Carbon incorporation into Si at high concentrations by ion implantation and solid phase epitaxy[END_REF]. In addition, 90 tiny carbon-enriched clusters of 2 to 3 nm diameters were found below the implantation peak. They precisely grow here because of the supersaturation of selfinterstitials that drives carbon diffusion and its precipitation [START_REF] Dumas | 3D atomic-scale investigation of carbon segregation in phosphorus co-implanted silicon[END_REF]. These clusters are not pure in carbon: they contained silicon atoms which are part of self-interstitials induced by ion implantation [START_REF] Cristiano | Interstitial trapping efficiency of c+ implanted into preamorphised silicon-control of EOR defects[END_REF][START_REF] Cacciato | Dislocation formation and B transient diffusion in C coimplanted Si[END_REF][START_REF] Goesele | Diffusion engineering by carbon in silicon[END_REF]. Self-interstitials are known to drive transient enhanced diffusion of dopants and the maturation of extended defects which are both to inhibit in the design of implanted-doped layers [START_REF] Minondo | The impact of the substrate preamorphisation on the electrical performances of p+/n silicon junction diodes[END_REF][START_REF] Cowern | Enhanced diffusion in silicon processing[END_REF]. That is why the atomic fraction of carbon in these clusters is of interest for the design of devices as it gives the number of trapped self-interstitials per carbon atom. In other works [START_REF] Cristiano | Interstitial trapping efficiency of c+ implanted into preamorphised silicon-control of EOR defects[END_REF][START_REF] Cacciato | Dislocation formation and B transient diffusion in C coimplanted Si[END_REF], the trapping ratio is found close to one, in agreement with the expected stoichiometry of the SiC phase present in the phase diagram [START_REF] Olesinski | The C-Si (carbon-silicon) system[END_REF]. However, in a recent APT study, the atomic fraction was found well below that of the SiC phase (2.5 at. % against 50 at. %) [START_REF] Dumas | 3D atomic-scale investigation of carbon segregation in phosphorus co-implanted silicon[END_REF]. This value was obtained from the distribution of distances between 1NN carbon events. As this method disregards the number of carbon atoms in molecular ions, it is now clear that it leads to an underestimation of the carbon amount.

Fig. 6. 3D distribution of carbon ions along the implantation axis after cluster identification (obtained from the lowest electric field analysis). 90 carbon enriched-clusters of average radii 2.5 +/-0.5 nm were found below the implantation peak. This corresponds to a cluster volume fraction of 0.0046+/-0.0003.

The average composition of carbon-enriched clusters shown in Fig. 6 was deduced from an erosion profile [START_REF] Vaumousse | A procedure for quantification of precipitate microstructures from three-dimensional atom probe data[END_REF] (Fig. 7). Away from the interface, composition is that of the matrix. The existence of a plateau in both profiles demonstrate that the core of the carbon-enriched zone is not affected by local magnification effects and related trajectory overlaps [START_REF] Blavette | A model accounting for spatial overlaps in 3D atom-probe microscopy[END_REF]. This makes it possible to derive a core composition close to 27 at. % at high field and 46 at. % at low field. This clearly show that the atomic fraction of carbon approaches the expected stoichiometry (50 at. %) only in low electric field conditions. This agrees with what was shown for the overall carbon level (Fig. 5).

-1,0 -0,5 0,0 0,5 1,0

Conclusion

Atom Probe Tomography was employed to quantify carbon in implanted silicon at two various electric fields (~ 15 and 20 V/nm). Most peaks were related to molecular ions originating from carbon-enriched clusters that nucleate due to carbon supersaturation.

Statistics and equal proportions of implanted 12 C and 13 C were successfully used to identify peaks and quantify the amount of carbon. Every mass-to-charge overlap was indeed resolved. We now know that (C2) 2+ ions are absent for electric fields close to those used in the present study. (C3) + family contribution to the carbon content was derived despite the presence of ((H2O)2) + ions.

It has been also shown that molecular ions significantly contribute to the carbon content. The fact that this contribution decreases when the electric field increases was attributed to the promotion of molecular ion dissociations close to the tip surface at higher electric fields. This in turn lead to a much more accurate carbon quantification using low electric fields (less multievents and consequently less detection biases).

Finally, the atomic fraction of carbon in clusters was derived from the value of the plateau in erosion profiles and was found close to that of the expected SiC phase when using low electric field conditions (high laser power).

Fig. 1 .

 1 Fig. 1. Scanning electron microscopy images of the tips before APT analysis a) tip analyzed at the highest electric field (tip diameter : 38 +/-3 nm ) b) tip analyzed at the lowest electric field (tip diameter : 28 +/-3 nm).

Fig . 3 .

 3 Fig .3. Mass spectra related to the filtered ions from the cluster identification (lowest electric field analysis). Peaks are identified by family in the range a) 0 to 28 Da b) 28 to 48 Da c) 48 to 74 Da.

Fig. 4 .

 4 Fig. 4. Histogram of the families respective contribution to the carbon content in both analyses.Contribution are sorted in mass-to-charge ratio increasing order.

Fig . 5 .

 5 Fig .5. APT and SIMS carbon concentration profiles with implantation depth. Error bars delimits the uncertainty window derived from counting statistics and background subtraction. LEF and HEF respectively stand for Lowest Electric Field and Highest Electric Field.

Fig. 7 .

 7 Fig. 7. Erosion profile of the carbon composition in carbon-enriched clusters. Error bars delimits the uncertainty window derived from counting statistics.

Table 1

 1

	).

Table 1 :

 1 Ions belonging to the (SiC) + family and their respective proportion of counts (lowest electric field analysis).

Acknowledgment:

The authors thank the STMicrolectronics Crolles Thermal Treatment Team for the design of a relevant annealing inducing carbon clustering and F.Verpilllot for the helpful discussions regarding molecular ion dissociation.


Charles Egloff
email: charles.egloff@gmail.com

J Sibiude

C Couffignal

L Mandelbrot

O Picone

Causes and consequences of fever during pregnancy: A retrospective study

Keywords: fever, pregnancy, influenza, etiology, complications, antibiotics

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Fever is one of the most frequent reasons for emergency consultation during pregnancy and may be associated with significant adverse outcomes, these being maternal (sepsis, organ damages) obstetrical (miscarriage, preterm birth, chorioamnionitis) or fetal (malformations, fetal demise).

However, only one study including mainly second and third trimester pregnant women, evaluated causes of acute undifferentiated fever and 25% of women had no identified cause. (1) Moreover, nearly 12% (IC95 8.6-16.8) of patients with fever during pregnancy required hospitalization in intensive care unit and it has been shown that bacteremia is complicated by fetal loss in 10% of cases. [START_REF] Surgers | Evolving microbiological epidemiology and high fetal mortality in 135 cases of bacteremia during pregnancy and postpartum[END_REF][START_REF] Knowles | Maternal sepsis incidence, aetiology and outcome for mother and fetus: a prospective study[END_REF] Published studies usually focus on a single etiology, such as influenza, or pyelonephritis, and some studies focus on fever as a symptom during labor. [START_REF] Mertz | Pregnancy as a risk factor for severe influenza infection: an individual participant data meta-analysis[END_REF][START_REF] Wing | Acute pyelonephritis in pregnancy: an 18-year retrospective analysis[END_REF][START_REF] Towers | Incidence of fever in labor and risk of neonatal sepsis[END_REF] To our knowledge, etiologies and complications of fever during pregnancy have never been studied.

There is no recommendation about fever in pregnant women, but usual care for undifferentiated fever is to introduce probabilistic antibiotic against listeria monocytogenes, responsible for overuse of antibiotics. Improving knowledge about etiology and management of fever in pregnant women could modify antibiotics prescription, which can have consequences on public health. [START_REF] Laxminarayan | Access to effective antimicrobials: a worldwide challenge[END_REF] The importance of correctly orienting diagnosis and care is underscored by the current COVID-19 epidemic.

The aim of this study was to determine etiology, antibiotics prescription and maternal or fetal complications in women consulting for fever during pregnancy.

METHODS

Study population:

We conducted a retrospective single center cohort study, including all pregnant women who presented for fever over one year. Inclusion criteria were: any pregnant women consulting between August 1, 2016 and July 31, 2017 to the gynecological emergency department of the Louis Mourier hospital (Assistance Publique des Hopitaux de Paris), a tertiary care center, with a temperature greater than or equal to 38°C (100.4°F) at home or at the emergency department. Among all consultations, use of antipyretics before the consultation could not be comprehensively collected. We chose to include all patients who had fever, even if fever was not confirmed in the Emergency room (ER). Because this group was usually not described in available literature, the population was then analyzed in two groups: patients whose fever was confirmed in the ER vs patients whose fever was not confirmed in the ER (fever only at home). Exclusion criteria were incomplete medical records, outcome of pregnancy not known or in progress at the time of the study and opposition by the patient to use of her medical data. We chose not to include pregnancy with unknown outcome in order to because the % of complications was an important outcome of our study. We wished to be the most exhaustive on outcome and avoid diagnostic errors.Clinical features, laboratory findings, prescription and outcomes were collected from electronic medical records (Diamm©, Villers les Nancy, France / Stare©, France / Carestream©, Rochester, United-Sate) with secure software (Redcap©, Vanderbilt University, Nashville, United State). Cold Season was defined as the period most sensitive for common viral infection, that is from October to March. ( 8 if lumbar pain, chest x-ray if chest pain or dyspnea). When fever is not confirmed at presentation, diagnostic testing is decided by the physician. Hospitalization was not systematic in our centre,but was largely carried out in case of confirmed fever without certainty of diagnosis, in order to monitor a possible Listeria. Similarly, antibiotic therapy against Listeria monocytogene was largely recommended in the absence of certainty of diagnosis. One diagnosis was assigned to every consultation, if two diagnoses were possible, the one with the higher probability was selected.

Statistical analysis and ethical committee

Continuous data were presented as medians and 25th to 75th percentiles. Categorical data were presented as counts and percentages. For the description of outcomes, the 95% confidence interval was estimated. Fisher exact test or Chi-square were performed for the categorical data as appropriate. Statistical analysis was performed using R software (version 3.5.1)

This study was approved by the Institutional Review Board (IRB status number 00006477) number 2018-032.

RESULTS

Diagnoses

Over the study period of one year, 3315 patients were followed for their pregnancy, and 121 consulted for fever at home or at the emergency departments, i.e. 3.6% (95% confidence interval [3; 4.3]. For 21 of these patients, medical charts were considered as incomplete and were excluded (Figure 1). We compared characteristics of patients according to the confirmation of fever at presentation (N=45; 45%; 95%CI 35-55) vs fever reported only at home (N= 55 ; 55% 95%CI 45-65) (Figure 1).

Median age of patients was 30 years [IQR 26-32].

No patient had preexisting diabetes, immunodeficiency or immunosuppressive therapy and they were all seronegative for HIV, hepatitis C and syphilis. Most consultations took place during cold season (73% from october to march).

Characteristics did not differ between the two groups (Table 1) except for the period of consultation (winter was more frequent in the group with no confirmed fever, p<0.005).

Concerning the etiologies of fever, 42 patients (42% ; 95%CI 32-52) had a certain diagnosis: 21 influenza (21% ; 95%CI 13-29), 11 acute pyelonephritis (11% ; 95%CI 5-17), 5 intra uterine infection (5% ; 95%CI 1-9), 5 others (5% ; 95%CI 1-10) (detailed in table 2), and 58 patients (58% ; 95%CI 48-67) had only a uncertain diagnosis : 37 common viral infection (37% ; 95%CI 28-46), including 2 cases of rhinovirus diagnosed on multiplex PCR, 6 viral acute gastroenteritis (6% ; 95%CI 1-11) and 15 unknown (15% ; 95%CI 8-22). (Table 2). Certain diagnosis was more often reported in women with confirmed fever at presentation than among women who reported fever only at home (60% vs 27%, p=0.001). No case of listeria was diagnosed. Common viral infection are defined as association of fever with one or more flu-like symptoms (chills, headache, myalgia, asthenia, coughing ), whereas in case of no symptoms associated to fever, we classified as unknown diagnosis (N=15).

Intrauterine infections occurred in patients who all had premature rupture of membranes: three at term and one at 22WG in a context of known cervical incompetency. The last one occurred in a case of prolonged rupture of membranes: PPROM was diagnosed at 17 WG and intra uterine infection at 31WG. All patients were in labour except two, at term, for which induce of labor was necessary.

Hospitalization and complications

In the group with confirmed fever at presentation, 46% (21 of 45 patients; 95%CI 32-62) were hospitalized, for a median of 3 days [IQR 2-4] and 22% (10 of 45 patients; 95%CI 10-34) presented maternal or fetal complications (Table 3). The fetal complications were: 2 (4%) early miscarriage, 2 (4%) late miscarriage, 2 (4%) preterm delivery at 31WG and 26WG, 1 (2%) stillbirth. The maternal complications were: 1 (2%) hospitalization for painful uterine contractions without preterm labor, 3 (6%) severe sepsis, 1 additional patient (2%) who required hospitalization in intensive care unit.

Early miscarriage occurred at 11 and 6 WG, one with a common viral infection and the other with positive influenza PCR. Late miscarriage concerned two patients. The first occurred at 19 WG, following hospitalization for fever, contraction and bleeding related to a subchorionic hematoma. The other was the intra uterine infection at 22 WG cited above.

One preterm delivery was the intra uterine infection at 31 WG with PPROM cited above. The other preterm delivery occurred in a patient hospitalized at 26WG for bleeding and uterine contraction with a temperature of 38°C (100.4°F) without evidence for intra uterine infection. The patient underwent a cesarean section for non-reassuring fetal heart rate and spontaneous labor, two days after hospitalization. Bacteriologic sample after birth was negative.

The stillbirth occurred after voluntary intoxication with 10 intra vaginal misoprostol tablets at 21 WG. The patient presented a severe case of hyperthermia at 40.7°C (105.3°F). No other etiology for fever was found.

One patient required intensive care unit hospitalization because of herpetic meningitis. She consulted at 38WG with fever (38.6°C/101.5°F), chills, emesis, headache, and signs of confusion.

PCR HSV in the cerebrospinal fluid was positive. A caesarean section was decided at 38WG following clinical deterioration with hemodynamic instability. The patient delivered a 3200g male infant, PCR HSV on the newborn was negative and he had a normal evolution. Maternal hospitalization was complicated by pneumonia and she was able to leave the intensive care unit after 14 days.

Both hospitalizations and complications were less frequent in the group with no confirmed fever at presentation (Fisher's exact test p<0.001 and p= 0.01 respectively). Among them, 3 of 55 (5% ; 95%CI 0-11) had complications : miscarriage at 7 and 8 WG and a fetal demise at 15 WG with no other cause. The patient consulted for fever at 38° at home, not confirmed at presentation, associated with headache and chest pain. Induction of labor was performed, and she delivered without complications or fever; the fetus had no apparent malformation. Among patients with no fever confirmed, 4 out of 55 (7%; 95%CI 0.2-14) were hospitalized, for a median of 2,5 days [IQR 1.75-3].

We also compared the rate of hospitalization and complications according to the fact that diagnosis was certain or not. Hospitalization rate was lower among patients for which the diagnosis was uncertain, 7/58 (12%; 95%CI 4-20) than among patients for which diagnosis was certain : 18/42 hospitalizations, 43%, p<0.001). Rate of complication did not differ : 8/58 (14% (95%CI 5-22) for uncertain diagnosis vs 5/42 complications, 12%, for patients with certainty of diagnosis p= 1.0).

Biological results

Among all patients, 43% (43/100) had blood exams with C-reactive protein (CRP), 29/45 of women with fever in the ER and 14/55 of women with fever only at home. CRP levels was not significantly different in the group with complications than in the group without complications.

(29.7mg/L vs 45.8 mg/L, p=0.221). (Figure 2). Moreover, 25% patients (2/8) in the group with complications had negative CRP (<6mg/L) and 9% patients (3/35) had negative CRP in the group without complications. The negative predictive value of a positive CRP for an adverse outcome was 60%.

Among all patients, 58% (58/100) had blood exams with white count cell (WCC), 37/45 of women with fever in the ER and 21/55 of women with fever only at home. WCC levels in the group with complications was significantly higher than in the group without complications. (14070x10 9 /L vs 10720x10 9 /L [IQR 8100; 13063] p=0.03). (Figure 2)

Antibiotics prescription and maternal treatment

Among all included women, 45% (45/100) received antibiotics and 29% (29/100) received antibiotics targeting listeria (amoxicillin 2g x 3 per days until result of blood cultures). Intra uterine infection and acute pyelonephritis were treated with appropriate antibiotics. Among 21 patients with influenza (PCR test positive), 15 received oseltamivir (75mg x 2 per day for five days) and the other six patients did not receive oseltamivir, because symptoms occurred more than 48 hours before the consultation. Three additional patients received oseltamivir although the PCR test was negative.

Among the 32 patients with confirmed fever at presentation and no acute pyelonephritis or intrauterine infection, probabilistic antibiotic against Listeria monocytogene was prescribed for 19 patients (59%, 19/32). No case of listeria was diagnosed. Prescription of antibiotics was more frequent among women with confirmed fever at presentation than among women who reported fever only at home (75% vs 20%, p < 0.001).

DISCUSSION

Although fever in pregnancy is a common clinical issue, this is the first study to estimate the prevalence of different etiologies and to describe management and complications. The diagnosis remained uncertain in the majority of cases after the initial work-up (58%). When fever was confirmed at presentation, 22% of developed a maternal or fetal complication. When the diagnosis was uncertain at the end of the examination, the majority (59%) received antibiotic therapy targeting Listeria monocytogenes. Uncertain diagnosis was not associated with length of hospital stay or a higher rate of complications. The large proportion of complications can probably be explained by the fact that patients consulting at the hospital emergency department are probably the most severe cases. Although advice is given to patients to come to the emergency department in case of temperature > 38°C, it is likely that some of the least severe patients may have consulted their primary physicians first, and our study may thus be biased towards the most severe patients. Incidence of fever during pregnancy (i.e.

3.6%

) is probable underestimated, because some patients may have consulted elsewhere, or not.

Influenza was one of the most frequent etiologies in our series. The influenza test was generally performed using specific PCR, but in five cases, multiplex PCR was performed, detecting two positive cases for rhinoviruses. Two retrospective studies conducted during the 2009-2010 flu pandemic found in pregnant women with influenza-like symptoms, non-influenza respiratory viruses rates of 23%, and influenza H1N1 rates between 31 and 42%, similar to our results. [START_REF] Pilorgé | Rhinoviruses as an underestimated cause of influenza-like illness in pregnancy during the 2009-2010 influenza pandemic[END_REF][START_REF] Paño-Pardo | Influenza-like illness in pregnant women during summertime: clinical, epidemiological and microbiological features[END_REF]. The multiplex PCR was not used on a routine basis because it is expensive and time-consuming, although this strategy could possibly avoid use of antibiotics and hospitalizations. Some studies have evaluated clinical and economic impact of multiplex respiratory virus assays, with different results, but this has never been assessed in a pregnant population [START_REF] Vallières | Clinical and economical impact of multiplex respiratory virus assays[END_REF]. Another alternative to the standard real-time influenza PCR for wich results is often delivered after several hours is to use a rapid molecular assay.

A recent study highlight benefit on hospitalization and antibiotics consumption, but no economic impact was done. (13) Some other novel biological tests, such as RNA biosignature are promising, but need to be validated. [START_REF] Mahajan | Association of RNA Biosignatures With Bacterial Infections in Febrile Infants Aged 60 Days or Younger[END_REF] In this study, CRP seems not to be predictor for adverse outcome, unlike white count cell. However, the low number of patients and the retrospective design of our study requires careful interpretation of this results. Although there is a protocol for the management of fever in our emergency department, this one was not always respected. For example, CRP and hemogram was performed in only 58% of the consultations and enterovirus PCR was never prescribed, although we now recommend performing it systematically in view of the fetal and neonatal risks reported recently. [START_REF] Méreaux | Enterovirus infection during pregnancy: Underestimated cause of fetal and neonatal complications?[END_REF] The current COVID-19 pandemic demonstrates performing a rapid diagnosis in pregnant women presenting with fever allow for appropriate care, including hospitalization vs. outpatient management and whether or not to prescribe antibiotics.

Our study demonstrates that fever during pregnancy is associated with a high rate of adverse events. The main strength of this study is the inclusion of all patients consulting for fever, and therefore including those where diagnosis was not certain. There is no answer available for patients who reported fever only at home, even though clinicians are regularly face to this situation. The analysis of this population in our studies is an original approach. These patients are mostly excluded from other studies, although etiology or inflammation associated with fever may have consequences for them as well. [START_REF] Kalagiri | Inflammation in Complicated Pregnancy and Its Outcome[END_REF][START_REF] Ashwal | Intrapartum fever and the risk for perinatal complications -the effect of fever duration and positive cultures[END_REF]. In this subgroup, we found a significant rate of complications (14%), one half of the total complications, even if complications are probably most often the result of the etiology or inflammation, and not fever per se. Because of this result, not shown until now, it seems necessary to conduct further prospective studies evaluating the rate of complication in case of fever, even if in cases where diagnosis is unclear. The complication rate was lower when fever was not confirmed, possibly related to less severe infections and in an unknown proportion an overestimation of the temperature when taken at home. Management of pregnant women with fever at home but not confirmed at presentation has not been described previously.

Management of fever during pregnancy, particularly the rate of antibiotic prescription, has never been evaluated, unlike in other populations (emergency department for adult or pediatric population), although administration of an antibiotic is much higher in pregnant women than in these populations. Beyond the allergic risk, cost-efficacy, and the issue of antibiotic resistance, recent data suggests that antibiotic therapy during pregnancy could influence the microbiota of newborn and influence risk of diabetes and obesity. [START_REF] Laxminarayan | Access to effective antimicrobials: a worldwide challenge[END_REF] [START_REF] Stoll | Changes in pathogens causing early-onset sepsis in very-low-birth-weight infants[END_REF] [START_REF] Jauréguy | Effects of intrapartum penicillin prophylaxis on intestinal bacterial colonization in infants[END_REF][START_REF] Kuperman | Antibiotic use during pregnancy: how bad is it?[END_REF]. Most patients (58%) have an uncertain diagnosis at the end of the consultation, and listeriosis is considered, given the high risk of complications including fetal loss. This may lead to excessive prescription of antibiotics despite the rarity of the diagnosis (about 30 cases per year in France among 800 000 births). In the study conducted by Charlier et al. out of 107 cases of fetal infection with Listeria monocytogenes, only 5% of patients had an isolated fever. [START_REF] Charlier | Clinical features and prognostic factors of listeriosis: the MONALISA national prospective cohort study[END_REF] We believe that developing more reliable diagnostic and prognostic strategies would reduce unnecessary hospitalizations, and unnecessary antibiotic prescriptions and thus economic costs, while being more vigilant when there is a significant risk of complications.

The limitations of this study are the size of our population and its retrospective observational nature. There are no confirmed criteria to predict a complication when a pregnant woman presents with fever, except according to specific diagnoses such as intrauterine infection. The low size of our population does not allow us to make this type of prediction. As our series was retrospective, some data may be lacking. All women with a temperature of 38°C or more at presentation were identified.

However, fever before presentation may be under-reported. Also, while all drug prescriptions were recorded, actual antibiotic consumption, especially concerning duration of the treatment, may have been variable depending on patients' compliance. Classifications of etiologies was done by several observers, but in some cases symptoms was unusual and not systematically confirm with microbiological or histologic investigations. As example, only the late miscarriage had placental analysis, which confirm histologic intra uterine infection. Unfortunately, for other patients, placental analysis was not available.

CONCLUSION

Fever during pregnancy was associated with a high rate of complications, 22% overall.

Further, prospective studies on larger cohorts, should be conducted to clarify the medical and public health consequences, including in the longer term, identify prognostic factors and allow for the study of new strategies.

) 2 . 2

 22 Variables collected: Determination of etiology. The management of fever during pregnancy in our gynecological emergency department consists in performing systematically a first line biological work-up including blood tests with hemogram, plasma C-reactive protein (CRP), and bacteriological samples with urine culture (UC), PCR enterovirus in the blood, vaginal swab and hemocultures for Listeria Monocytogene. Second line examination depends on maternal symptoms (ex: PCR influenza if flu-like syndrome, renal ultrasound

2. 3 -

 3 Categorization of etiologies: Etiologies were divided in two different group, according the certainty of diagnosis. Diagnosis criteria were decided by CE and OP. All etiologies were than classified by CE. In the rare cases of difficulty in classifying etiologies, cases were reviewed by JS. • Certainty of diagnosis -Intra uterine infection: fever in the context of premature rupture of membranes, associated with at least one supplementary criteria among the following : persistent fetal tachycardia, painful uterine contractions, spontaneous labor or purulent amniotic fluid defined as the French guidelines.(9) Acute pyelonephritis: lumbar pain and positive urine culture without other most likely diagnosis -Influenza: nasopharyngeal PCR positive for influenza A/B -Listeriosis: Positive hemoculture for Listeria monocytogene -Other: proof of diagnosis explaining the fever, with etiology not classifiable in categories mentioned above • Probability of diagnosis (only in the absence of a certain diagnosis) -Common viral infection: Flu-like symptoms ie. chills, headache, myalgia, asthenia, coughing without other most likely diagnosis -Viral acute gastroenteritis: compatible symptoms (intestinal disorder, nausea/vomiting, abdominal pain) without any other most likely diagnosis -Unknown: no etiology found, eg. no symptoms or work-up compatible with any of the diagnoses cited above.

Figure

  Figure 1. Flow chart. Women presenting for fever in pregnancy in the emergency department between

Table 1 .

 1 1. Flow chart. Women presenting for fever in pregnancy in the emergency department between 08/01/2016 and 07/31/2017.Figure 2.Comparative level of C-reactive protein (CRP) in mg/L and white count cells at the time of consultation for fever in the group with complications of pregnancy and the group without complications. Characteristic of the 100 included pregnant women according the fever status at presentation (confirmed vs only at home).

	121 pregnant patients
	consulting for fever
		21 records with lack
		of data
	100 patients included
	45 patients with confirmed fever at presentation	55 patients with fever only at home
	10 influenza	11 influenza
	8 acute pyelonephritis	3 acute pyelonephritis
	5 intra uterine infection	0 Intra uterine infection
	4 others	1 other
	11 common viral infection	26 common viral infection
	0 viral acute gastroenteritis	6 viral acute gastroenteritis
	7 unknown diagnosis	8 unknown diagnosis