Marina Chauvet

Arthur Monjot

Anne Moné

Cécile Lepère
email: cecile.lepere@uca.fr

Single cell analysis reveals a new Microsporidia-host association in a freshwater lake

Keywords: Microsporidia, host-parasite association, single cell, TSA-FISH, lake, microbial ecology

Microsporidia are a large group of obligate intracellular eukaryotic parasites. Recent studies suggest that their diversity can be huge in freshwater lake ecosystems especially in the <150 µm size fraction. However, little is known about their hosts and therefore their impact on the trophic food web functioning. In this study, single cell analysis and fluorescence microscopy were used to detect new host-parasite association within rotifer communities in lake Aydat (France). Our analysis showed the existence of a potential new species belonging to the Crispospora genus able of infecting the rotifer Kellicottia with a high prevalence (42.5%) suggesting that Microsporidia could have a great impact on the rotifer populations regulation in lakes.

Microsporidia are obligate intracellular eukaryotic parasites related to Fungi. They are mostly known to parasitize a broad range of hosts among the animal kingdom in different environments [START_REF] Murareanu | Generation of a Microsporidia Species Attribute Database and Analysis of the Extensive Ecological and Phenotypic Diversity of Microsporidia[END_REF]. Recent metabarcoding studies revealed an unexpected diversity in lake ecosystems especially in the <150 µm size fraction suggesting that Microsporidia could have an impact on the planktonic communities at the base of the food web [START_REF] Dubuffet | A phylogenetic framework to investigate the microsporidian communities through metabarcoding and its application to lake ecosystems[END_REF][START_REF] Chauvet | Temporal variations of Microsporidia diversity and discovery of new host-parasite interactions in a lake ecosystem[END_REF]. However, they have rarely been described as parasites of protists and microzooplankton. In marine environments, they are mainly known to parasite gregarines (e.g. [START_REF] Mikhailov | Genomic Survey of a Hyperparasitic Microsporidian Amphiamblys sp. (Metchnikovellidae)[END_REF]), but they were also shown in association with Paramyxea [START_REF] Stentiford | Hyperspora aquatica n.gn., n.sp. (Microsporidia), hyperparasitic in Marteilia cochillia (Paramyxida), is closely related to crustacean-infecting microspordian taxa[END_REF]. In freshwaters, only a few observations showed Microsporidia parasiting ciliates with only two microsporidian species characterized by morphological and molecular approaches (Euplotespora binucleata and Globosporidium paramecii parasitizing the ciliates Euplotes woodruffi and Paramecium aurelia, respectively [START_REF] Fokin | Euplotespora binucleata n. gen., n. sp. (Protozoa: Microsporidia), a parasite infecting the hypotrichous ciliate Euplotes woodruffi, with observations on microsporidian infections in ciliophora[END_REF][START_REF] Yakovleva | The first case of microsporidiosis in Paramecium[END_REF]). Among microzooplankton, Microsporidia have been observed in the rotifers Brachionus calicyflorus as well as in Asplanchna priodonta and A. brightwellii in both river and freshwater lakes [START_REF] Gorbunov | Parasites in rotifers from the Volga delta[END_REF][START_REF] Wolska | Parasites of zooplankton and periphyton assemblages in the littoral zone of lakes in Drawa National Park, Poland[END_REF] however these observations (no molecular data) remain sporadic and for most at low prevalence.

Moreover, with co-occurrence network and microscopic observations, a more recent study suggested that Microsporidia could infect a broader range of rotifers [START_REF] Chauvet | Temporal variations of Microsporidia diversity and discovery of new host-parasite interactions in a lake ecosystem[END_REF]. We therefore used, in this study, single cell analysis as well as Tyramide Signal Amplification -Fluorescent In Situ Hybridization (TSA-FISH) to detect new host-parasite association within microzooplankton communities in a lake ecosystem (lake Aydat, Massif Central, France) and especially (i) confirm the hypothesis raised about the broader range of rotifers parasitized by Microsporidia and (ii) characterize by molecular information and microscopic observation both partners.

Zooplankton was sampled with vertical tows of 20 µm-mesh plankton net at the central point of the lake (depth = 14 m) on the 18/05/2021 (see Supplementary Material). 17 rotifers, identified morphologically as probably belonging to the genera Kellicottia (n = 13) and Keratella (n = 4), were isolated by micromanipulation and individually placed in a 96 wells plate before Whole Genome Amplification (WGA) (see Supplementary Material). The Single Amplified Genomes (SAGs) were then screened for the presence of Microsporidia by the amplification of the V1-V3 region of the microsporidian gene coding for the SSU rRNA according to Chauvet et al. [START_REF] Chauvet | Temporal variations of Microsporidia diversity and discovery of new host-parasite interactions in a lake ecosystem[END_REF]. The sequences obtained after cloning and sequencing were compared to those available in public databases and included in a phylogenetic tree (see Supplementary Material). This analysis showed that on the 17 isolated rotifers, 6 Kellicottia had a positive PCR amplification signal with Microsporidia primers however no Microsporidia amplification was recorded in Keratella. All Microsporidia sequences were affiliated to the genus Crispospora (Fig. 1) which is affiliated to the clade IV2; Arlequin group (defined in Dubuffet et al. [START_REF] Dubuffet | A phylogenetic framework to investigate the microsporidian communities through metabarcoding and its application to lake ecosystems[END_REF]), and well represented (~ 50% of the total OTUs) in metabarcoding studies conducted in lake Aydat [START_REF] Dubuffet | A phylogenetic framework to investigate the microsporidian communities through metabarcoding and its application to lake ecosystems[END_REF][START_REF] Chauvet | Temporal variations of Microsporidia diversity and discovery of new host-parasite interactions in a lake ecosystem[END_REF]. With 97% identity with Crispospora chironomi (the only known species of the genus so far) these sequences could represent a new species of the Crispospora genus (Fig. S2a). Indeed, although thresholds for species delineation may vary, an identity close to 100% is suggested for taxa separation at the species level for Microsporidia [START_REF] Dubuffet | A phylogenetic framework to investigate the microsporidian communities through metabarcoding and its application to lake ecosystems[END_REF][START_REF] Tokarev | A formal redefinition of the genera Nosema and Vairimorpha (Microsporidia: Nosematidae) and reassignment of species based on molecular phylogenetics[END_REF].

Crispospora chironomi is known to parasite chironomids eggs [START_REF] Tokarev | Life cycle, ultrastructure, and molecular phylogeny of Crispospora chironomi g.n. sp.n. (Microsporidia: Terresporidia), a parasite of Chironomus plumosus L. (Diptera: Chironomidae)[END_REF] that can be found in aquatic environments especially when they are eutrophic and anthropized [START_REF] Langdon | Assessing lake eutrophication using chironomids: understanding the nature of community response in different lake types[END_REF].

On the Microsporidia positive SAGs, rotifers were characterized by amplification of the COI using the 30F and 885R primers set according to Zhang et al. [START_REF] Zhang | A new set of highly efficient primers for COI amplification in rotifers[END_REF] with some modifications (see Supplementary Material). The rotifer affiliation analysis revealed that this potential new species of Crispospora is able to parasite the genus Kellicottia (Fig. S1). Even though Kellicottia sequences grouped into two clades in the phylogenetic analysis, species determination is challenging due to high intraspecific variation of identity percent which can be higher than 20% for rotifers (Fig. S2b) [START_REF] Zhang | Application of COI Primers 30F/885R in Rotifers to Regional Species Diversity in (Sub)Tropical China[END_REF].

To visualize and quantify Microsporidia-rotifer associations, TSA-FISH was performed on the rest of the plankton net sample, using a Microsporidia specific probe (USP01) according to Chauvet et al. [START_REF] Chauvet | Temporal variations of Microsporidia diversity and discovery of new host-parasite interactions in a lake ecosystem[END_REF] (see Supplementary Material). Positive signals were obtained in Kellicottia rotifers with a surprisingly high prevalence (42.5%, total rotifers observed = 73) (Fig. 2).

Microsporidia were so far recorded in a small number of rotifers [START_REF] Gorbunov | Parasites in rotifers from the Volga delta[END_REF][START_REF] Wolska | Parasites of zooplankton and periphyton assemblages in the littoral zone of lakes in Drawa National Park, Poland[END_REF]. Microsporidium sp. and Microsporidium asperospora have been observed in Brachionus calicyflorus, and Microsporidium polygona in Asplanchna priodonta and A. brightwellii in freshwater ecosystems most of the time at low prevalence (e.g. Brachionus calyciflorus by Microsporidium sp. on 1.2% of the host population, [START_REF] Wolska | Parasites of zooplankton and periphyton assemblages in the littoral zone of lakes in Drawa National Park, Poland[END_REF]). However, these descriptions were only morphological and neither the site of infection nor the microsporidian spore or intracellular form were described. Here, we identified an infection which could be in the rotifer's stomach or pseudocoelom. The intracellular forms of the Microsporidia were on average 1.53 μm long, most being oval, some spherical. These findings were consistent with the described shape of the only known Crispospora species (i.e. Crispospora chironomi), with two shapes identified: short oval and spherical, with a size of 1.5 x 2.5 μm and 1.5 x 2 μm, respectively [START_REF] Tokarev | Life cycle, ultrastructure, and molecular phylogeny of Crispospora chironomi g.n. sp.n. (Microsporidia: Terresporidia), a parasite of Chironomus plumosus L. (Diptera: Chironomidae)[END_REF]. This finding is highly relevant since Microsporidia, along with hyphomycetes and oomycetes [START_REF] Barron | A New Genus of the Hyphomycetes Endoparasitic in Rotifers[END_REF][START_REF] Thomas | The ecology and phylogeny of oomycete infections in Asplanchna rotifers: Oomycete infections in Asplanchna[END_REF] could be an important regulator of these populations in lakes. Rotifers are indeed an important component of plankton in aquatic ecosystems playing fundamental role in both microbial loop and classical food web and as a link in energy flow between primary producers (algae, cyanobacteria) and higher trophic levels (zooplankton, shellfish, fish) [START_REF] Rico-Martínez | Rotifers as Models in Toxicity Screening of Chemicals and Environmental Samples[END_REF]. Kellicottia can reach 600 ind./l in lake Aydat and is the most abundant rotifers along with Keratella [START_REF] Lair | Grazing and assimilation rates of natural populations of planktonic rotifers Keratella cochlearis, Keratella quadrata and Kellicottia longispina in a eutrophic lake (Aydat, France)[END_REF].

Microsporidia sequences retrieved during this study had an identity >99% with an highly abundant OTU identified in the same lake in a previous study where the temporal dynamic of the Microsporidia diversity was analyzed over one year [START_REF] Chauvet | Temporal variations of Microsporidia diversity and discovery of new host-parasite interactions in a lake ecosystem[END_REF] (Fig. S2a). This Crispospora OTU and the only OTU affiliated to Kellicottia presented the same pattern of temporal variations (data not shown). This co-variation and the high prevalence of this association suggest an impact on the dynamic of Kellicottia in the lake however this needs to be confirmed for example We included in this phylogeny several sequences from undescribed microsporidian species (found through BLAST search) and a sequence found extensively in a previous study (in italic, [START_REF] Chauvet | Temporal variations of Microsporidia diversity and discovery of new host-parasite interactions in a lake ecosystem[END_REF]), in addition to described species, all belonging to the Arlequin group (clade IV2).

Fig. 1

 1 Fig. 1 Maximum Likelihood phylogeny of microsporidian sequences.

  Sequences from this study are given in bold. Outgroup species are representative species of other groups belonging to clade IV2. Numbers next to branches indicate the ML bootstrap support (100 replicates).

Fig. 2 FJ756125

 2 Fig. 2 Microsporidia (targeted by TSA-FISH with the USP01 probe, [3]) infecting a rotifer

Acknowledgements

The authors are very grateful to Jonathan Colombet and Eugénie Carriere for their help with sampling. We also gratefully acknowledge the platform CLIC (CLermont-ferrand Imagerie Confocale, Université Clermont Auvergne) and especially Caroline Vachias for her assistance with the confocal microscopy. The work of MC and AM was supported by two PhD fellowships funded by the 'Ministère de l'Enseignement Supérieur de la Recherche et de l'Innovation'.

Availability of data and material

All data and material are available in this publication and additional information online (supplementary material and supplementary figures). Sequences are available on GenBank, accession numbers: from ON026091 to ON026096 for the microsporidian sequences and from ON033621 to ON033624 for the rotifers.

by the culture of both partners which will also allow the characterization of the developmental cycle of this potential new species of Microsporidia.

Statements and Declarations

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing Interests

The authors declare they have no competing interests.


Tangui Barré

Patrick Mercie

Fabienne Marcellin
email: fabienne.marcellin@inserm.fr

Laure Esterle

Claudine Duvivier

Elina Teicher

Morgane Bureau

Julie Chas

Dominique Salmon-Ceron

Philippe Sogni

Patrick Mercié

Dominique Salmon-Céron

Maria Patrizia Carrieri

Linda Wittkop

Camelia Protopopescu

Anrs Co13

Hepavih Study Group

P Trimoulet

J Izopet

L Serfaty

V Paradis

B Spire

P Carrieri

M A Valantin

G Pialoux

I Poizot-Martin

K Barange

A Naqvi

E Rosenthal

A Bicart-See

O Bouchaud

A Gervais

C Lascoux-Combe

C Goujard

K Lacombe

D Neau

M Lack

S Maynard

M Radenne

F Amiri

; Valour

J Hépato-Gastro-Entérologie :

F Koffi

F Zoulim

P Bailly

C Radenne

C Augustin-Normand

T T Scholtes

Chu Le-Thi

Dijon Dijon

: L Département D'infectiologie

P Piroth

M Chavanet

Van Duong

M Huyen

A Buisson

S Waldner-Combernoux

A Salmon Mahy

C Rousseau

C H Martins

Perpignan Perpignan

H Aumaître

S Galim

Robert Chu

Debré

F Bani-Sadr

D Lambert

Y Nguyen

J L Berger

M Hentzien

V Brodard

Strasbourg ( Chru

D Le Trait D'

M Rey

M L Partisani

C Batard

M Cheneau

C Priester

E Bernard-Henry

P De Mautort

Virologie : P Gantner S Fischer

Fafi-Kremer

F Roustant

P Platterier

I Kmiec

L Traore

S Lepuil

S Parlier

V Sicart- Payssan

E Bedel

S Anriamiandrisoa

C Pomes

F Touam

C Louisin

M Mole

C Bolliot

P Catalan

M Mebarki

A Adda-Lievin

P Thilbaut

Y Ousidhoum

F Z Makhoukhi

O Braik

R Bayoud

C Gatey

M P Pietri

V Le Baut

R Ben Rayana

D Bornarel

C Chesnel

D Beniken

M Pauchard

S Akel

S Caldato

C Lions

A Ivanova

A-S Ritleg

C Debreux

L Chalal

J Zelie

H Hue

A Soria

M Cavellec

S Breau

A Joulie

P Fisher

S Gohier

D Croisier-Bertin

S Ogoudjobi

C Brochier

V Thoirain-Galvan

M Le

Cam Management

M Chalouni

V Conte

L Dequae-Merchadou

M Desvallees

C Gilbert

S Gillet

R Knight

T Lemboub

L Michel

M Mora

P Roux

S Tezkratt

T Rojas Rojas

M Baudoin

M Santos

V Di Beo

M Nishimwe

HCV Cure and Cannabis Abstinence Facilitate Tobacco Smoking Quit Attempts in HIV-HCV Co-Infected Patients (ANRS CO13 HEPAVIH Cohort Study)

Keywords: cannabis, tobacco, smoking cessation, human immunodeficiency virus, hepatitis C, chronic

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

Introduction

Tobacco smoking prevalence among people living with HIV (PLWH) is over 50% in high-income Western countries [START_REF] Park | Prevalence of non-HIV cancer risk factors in persons living with HIV/AIDS: a meta-analysis[END_REF]. In France, it was estimated at 37.5% in a national representative survey of PLWH conducted in 2011, with variations according to both sex and epidemiological group (from 7.9% among female migrants from Sub-Saharan Africa to 74.3% and 79.9% for past or current male and female injecting drug users (IDU), respectively) [START_REF] Tron | ANRS-Vespa2 study group. Tobacco smoking in HIV-infected versus general population in france: heterogeneity across the various groups of people living with HIV[END_REF]. More recent hospital-based data (FHDH-ANRS CO4, 2014) showed a tobacco smoking prevalence of 43.2% among French PLWH (3), which is nearly twice that observed in the French general population (4). This high prevalence could be explained by several factors including socioeconomically deprived status, stress, depression, other substance use and poor access to tobacco cessation treatment [START_REF] Reynolds | HIV: MORE EVIDENCE FOR ACTION[END_REF], as well as an overrepresentation of men who have sex with men (MSM) and IDU, two populations with a high prevalence of tobacco smoking, irrespective of HIV status [START_REF] Marshall | Tobacco use and nicotine dependence among HIV-infected and uninfected injection drug users[END_REF][START_REF] Mckirnan | Elevated risk for tobacco use among men who have sex with men is mediated by demographic and psychosocial variables[END_REF]. Hepatitis C virus (HCV)-infected individuals are also more likely to smoke tobacco. A cohort representative of HCV-infected persons in France highlighted an estimated tobacco smoking prevalence of 62% [START_REF] Barré | Cannabis use is associated with a lower risk of diabetes in chronic hepatitis C-infected patients (ANRS CO22 Hepather cohort)[END_REF], while in the United States, the estimated prevalence in this population was three times higher than in HCV-uninfected people [START_REF] Kim | Cigarette Smoking in Persons Living with Hepatitis C: The National Health and Nutrition Examination Survey (NHANES), 1999-2014[END_REF]. Similarly, tobacco smoking is very prevalent in HIV-HCV co-infected people [START_REF] Chew | Short Communication: Coronary Heart Disease Risk by Framingham Risk Score in Hepatitis C and HIV/Hepatitis C-Coinfected Persons[END_REF][START_REF] Knight | Significant reductions in alcohol use after Hepatitis C treatment: Results from the French ANRS CO13-HEPAVIH cohort: Significant Reductions in Alcohol Use[END_REF], a population prone to both depression [START_REF] Fialho | Depression in HIV and HCV co-infected patients: a systematic review and meta-analysis[END_REF] and psychoactive substance use [START_REF] Cacoub | Burden of HIV and hepatitis C co-infection: the changing epidemiology of hepatitis C in HIV-infected patients in France[END_REF][START_REF] Hagan | HIV/hepatitis C virus co-infection in drug users: risk behavior and prevention[END_REF], two known risk factors of low success rates in smoking cessation in PLWH [START_REF] Kariuki | HIV and smoking: associated risks and prevention strategies[END_REF]. Thanks to highly active antiretroviral therapy (ART), the life expectancy of PLWH has dramatically increased in recent decades [START_REF]Life expectancy of individuals on combination antiretroviral therapy in high-income countries: a collaborative analysis of 14 cohort studies[END_REF], while AIDS-related mortality has decreased [START_REF] Morlat | Causes of death among HIV-infected patients in France in 2010 (national survey): trends since 2000[END_REF]. Today, cancer and cardiovascular diseases are two leading causes of death in this population. ART-treated PLWH who smoke tobacco may now lose more life-years through smoking than through HIV infection [START_REF] Helleberg | Smoking and life expectancy among HIV-infected individuals on antiretroviral therapy in Europe and North America[END_REF].

Similarly, thanks to the scaling up of direct-acting antiviral treatment against HCV, it is expected that the related decrease in mortality in HCV mono-infected patients and HIV-HCV co-infected patients will be partially offset by tobacco-related mortality if nothing is done to reduce associated harms [START_REF] Gartner | Extending survival for people with hepatitis C using tobacco dependence treatment[END_REF][START_REF] Kronfli | Trends in causespecific mortality in HIV-hepatitis C coinfection following hepatitis C treatment scale-up[END_REF].

Engagement in HCV treatment has a positive impact on drug use and injection equipment sharing behaviors in IDU [START_REF] Caven | Impact of Hepatitis C treatment on behavioural change in relation to drug use in people who inject drugs: A systematic review[END_REF], but no such association with tobacco use has been found for HIV-HCV co-infected patients [START_REF] Yeung | Changes in quality of life, healthcare use, and substance use in HIV/hepatitis C coinfected patients after hepatitis C therapy: a prospective cohort study[END_REF]. Furthermore, data on changes in tobacco smoking behaviors following HCV cure in HIV-HCV co-infected patients are scarce.

Using data from the French National Agency for Research on Aids and Viral Hepatitis (ANRS) CO13 HEPAVIH cohort of HIV-HCV co-infected patients, we aimed to identify potential clinical (including HCV cure) and socio-behavioral correlates of tobacco smoking quit attempts (TSQA).

Materials and methods

Design of the cohort ANRS CO13 HEPAVIH is an ongoing French national multicenter prospective cohort of HIV-HCV co-infected patients initiated in 2005 (recruitment ended in 2016) which provides a representative sample of the French co-infected population receiving care in hospitals [START_REF] Loko | The French national prospective cohort of patients co-infected with HIV and HCV (ANRS CO13 HEPAVIH): early findings, 2006-2010[END_REF]. Only patients from the first recruitment phase (2005)(2006)(2007)(2008) were included in the present study population, as the content and timing of the cohort study's socio-behavioral questionnaires were subsequently modified. In the first phase, consecutive patients attending outpatient services in 17 different hospitals were enrolled according to the following selection criteria: aged ≥18 years; infected with HIV-1; either chronically co-infected with HCV (as confirmed by an HCV RNA assay) or HCV cured thanks to treatment. These participants had annual clinical follow-up visits (biannual (i.e., six-monthly) for patients with cirrhosis), with additional intermediate visits for patients initiating HCV treatment during follow-up.

Clinical data

Clinical, biological, and histological data were collected using standardized medical forms completed by medical staff at each clinical visit. Collected data included CD4-T cell count, plasma HIV RNA level, HIV transmission mode, time since HIV and HCV diagnosis, HCV treatment status and information on sustained virological response (HCV cure), serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, platelet counts, height and weight.

Socio-behavioral data

All patients (including cirrhotic patients) completed a self-administered questionnaire at enrolment and at each annual visit during the five-year follow-up. This questionnaire detailed sociodemographic characteristics (age, sex, living with a partner or not, employment status, perceived comfort of housing, educational level), psychosocial (depressive symptoms, previous treatment for depression, HIV-related self-reported symptoms) and behavioral data (alcohol and coffee consumption, and psychoactive substance use).

Tobacco smoking status

Tobacco smoking status was collected through a face-to-face interview with a physician at each clinical visit and categorized as a time-dependent variable with the following categories: 'non-smoker', 'former smoker', or 'current smoker'.

Study population

The present study used data from the five-year follow-up of HIV-HCV coinfected patients enrolled in the first phase of HEPAVIH cohort between 2005 and

2008.

The study population comprised patients who had at least one follow-up visit where they both reported being a current smoker and had available data from the self-administered questionnaire, and whose smoking status was available for at least one subsequent follow-up visit.

Study period

For each patient, the study period began at the first clinical visit where the patient reported being a current smoker and where a self-administered questionnaire was also available (hereafter 'baseline'). The study period ended at the first clinical visit where the participant reported being a former smoker (corresponding to a TSQA), or the last clinical visit with available tobacco smoking status during the follow-up period (hereafter 'end-of-study visit').

Outcome

The study outcome was time to the first TSQA during follow-up, defined as the time between baseline and the first transition from 'current smoker' to 'former smoker' status. Accordingly, duration was not considered when defining TSQA, and therefore TSQA included both temporary and definitive cessation events. As tobacco status was recorded annually (biannually for patients with cirrhosis), the study outcome was interval-censored.

Explanatory variables

All variables except sex, educational level and HIV transmission mode were time-dependent.

HCV cure was evaluated at each follow-up visit. Patients who had a 12-week sustained virological response at a given clinical follow-up visit, and those who cleared HCV before enrolment in the cohort, were classified as cured.

Given the potential therapeutic use of cannabis in the course of HIV and HCV infections [START_REF] Prentiss | Patterns of Marijuana Use among Patients with HIV/AIDS Followed in a Public Health Care Setting[END_REF][START_REF] Fischer | Treatment for hepatitis C virus and cannabis use in illicit drug user patients: implications and questions[END_REF][START_REF] Santos | HCV-Related Mortality Among HIV/HCV Co-infected Patients: The Importance of Behaviors in the HCV Cure Era (ANRS CO13 HEPAVIH Cohort)[END_REF] and the fact that its primary route of administration (i.e., smoking) is the same as that for tobacco [START_REF] Hindocha | No Smoke without Tobacco: A Global Overview of Cannabis and Tobacco Routes of Administration and Their Association with Intention to Quit[END_REF], cannabis use was tested separately from other psychoactive substances (grouped together in the analysis) as a potential correlate of TSQA. The frequency of recent cannabis use -understood here as during the previous four weeks -was categorized as 'never', 'sometimes', 'regular' or 'daily', with two variables tested as follows: never versus sometimes and more frequently (i.e., a dichotomous variable), and never / sometimes / regular or daily (i.e., a three-level variable). HIV transmission mode was categorized as 'IDU', 'MSM' and 'other'.

Recent use (i.e., in the previous four weeks) of psychoactive substances other than cannabis (cocaine, heroin, crack, ecstasy, street buprenorphine, amphetamines and LSD/other hallucinogens) was coded as 'yes' or 'no'.

Plasma HIV viral load was categorized as detectable versus undetectable, according to the limit of detection of the laboratory test used in each of the participating hospitals. Liver fibrosis was assessed using the FIB-4 index, which is a non-invasive marker of fibrosis calculated using age, AST level, ALT level, and platelet count with the following formula: age [years] * AST [IU/L])/(platelet count [10 9 /L] * (ALT [IU/L]) 1/2 . The presence of advanced fibrosis was defined as a FIB-4 index >3.25 [START_REF] Richard | Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection[END_REF]. Body mass index (BMI) was calculated as the weight Employment status was categorized as 'unemployed' versus all other situations (employed, retired, housewife/househusband). Perceived comfort of housing was derived from the question 'Would you say that your current housing is…?:' with 4 possible answers subsequently categorized as 'not comfortable' (which included the answers 'not at all comfortable' and 'barely comfortable') versus 'comfortable' (which included 'quite' and 'very comfortable'). This variable was designed and used as a proxy for standard of living. Educational level was categorized as having a high-school certificate or not. The presence of depressive symptoms was assessed using the CES-D scale (with cut-offs of 17 and 23 for men and women, respectively) [START_REF] Radloff | The CES-D Scale: A Self-Report Depression Scale for Research in the General Population[END_REF][START_REF] Fuhrer | La version française de l'échelle CES-D (Center for Epidemiologic Studies-Depression Scale). Description et traduction de l'échelle d'autoévaluation[END_REF]. The total number of HIV-related self-reported symptoms in the previous four weeks was based on the 39-item ANRS AC24 selfreported symptoms checklist [START_REF] Rosenthal | Efficacy, safety and patient-reported outcomes of ledipasvir/sofosbuvir in NS3/4A protease inhibitorexperienced individuals with hepatitis C virus genotype 1 and HIV coinfection with and without cirrhosis (ANRS HC31 SOFTRIH study)[END_REF], itself derived from the AIDS Clinical Trials Group (ACTG) Symptom Distress Module [START_REF] Ac | Development and Validation of a Self-Completed HIV Symptom Index[END_REF]. Alcohol consumption was assessed using the Alcohol Use Disorders Identification Test-Concise (AUDIT-C) questionnaire [START_REF] Bush | The AUDIT alcohol consumption questions (AUDIT-C): an effective brief screening test for problem drinking. Ambulatory Care Quality Improvement Project (ACQUIP). Alcohol Use Disorders Identification Test[END_REF], and categorized as hazardous (score ≥ 4 and ≥ 3 for men and women, respectively) or not. Coffee intake was categorized as daily (≥ 1 cup per day) versus non-daily.

Statistical analyses

We described the study population's main characteristics at the end-of-study visit, and the number of events occurring during the study period for each category of each explanatory variable.

For HEPAVIH participants (first phase of enrolment) who reported at least once during their follow-up that they currently smoked tobacco, we compared age and gender between included individuals (i.e., study population) and those excluded, using Mann-Whitney and chi-squared tests.

Associations between clinical (including HCV cure) and socio-behavioral characteristics and TSQA were assessed using a multivariable discrete-time proportional hazards Cox model with the exact partial-likelihood method to deal with tied failures. This type of model is recommended for survival analyses with intervalcensored data (34) -as was the case in the present study -where follow-up data are collected on an annual or biannual basis. Missing values for explanatory timedependent variables were imputed according to the last observation carried forward (LOCF) method. For each variable, this consisted in imputing each missing value with the last observed available value of the individual, if such a value existed. In order to minimize the risk of reverse causality, values at the n-1 visit for all time-dependent variables were used in the model to explain tobacco status at the following n visit.

Depressive symptoms and a history of treatment for depression were tested alternatively in the multivariable model.

Explanatory variables with a p-value < 0.20 (Wald test) in the univariable analyses were tested in the multivariable analysis. Variables were then selected using a stepwise backward procedure, based on the Wald test. Only those with a pvalue < 0.05 were retained in the final multivariable model.

The proportionality of hazards assumption was tested for all explanatory variables in the final multivariable Cox model using graphical examination of Kaplan-Meier estimates and statistical testing based on rescaled Schoenfeld residuals. We hypothesized that loss-to-follow-up (due to missed clinical visits, cohort attrition, and death) was non-informative (i.e., patients were lost to follow-up due to reasons unrelated to the study outcome, and therefore Cox model results were unbiased).

Stata/SE 14.2 software (StataCorp LP, College Station, TX) was used for all analyses.

Results

Characteristics of the study population

The study population comprised 695 participants (Figure 1), representing 59.1% of the individuals enrolled in the first phase of the HEPAVIH cohort, and 77.0% of the individuals who declared at least once during the follow-up that they currently smoked tobacco. Mann-Whitney and chi-squared tests revealed that that women accounted for a greater proportion of the participants, and that participants were slightly older than persons not included (data not shown). During the study period-which represented 2485 person-years -88 TSQA were observed, leading to an incidence rate of 3.5 TSQA/100 person-years (95% confidence interval (CI) [2.87-4.36]). The median follow-up time of study participants was 48 months (interquartile range (IQR) ).

The study population's characteristics at the end-of-study visit are provided in Table 1. Patients were mainly male (70.5%) and HIV-infected through drug injection (72.5%). Median age was 47.0 years (IQR [44.0-50.0]). Median time since HIV and HCV diagnosis was 21 (IQR [START_REF]Life expectancy of individuals on combination antiretroviral therapy in high-income countries: a collaborative analysis of 14 cohort studies[END_REF][START_REF] Morlat | Causes of death among HIV-infected patients in France in 2010 (national survey): trends since 2000[END_REF][START_REF] Helleberg | Smoking and life expectancy among HIV-infected individuals on antiretroviral therapy in Europe and North America[END_REF][START_REF] Gartner | Extending survival for people with hepatitis C using tobacco dependence treatment[END_REF][START_REF] Kronfli | Trends in causespecific mortality in HIV-hepatitis C coinfection following hepatitis C treatment scale-up[END_REF][START_REF] Caven | Impact of Hepatitis C treatment on behavioural change in relation to drug use in people who inject drugs: A systematic review[END_REF][START_REF] Yeung | Changes in quality of life, healthcare use, and substance use in HIV/hepatitis C coinfected patients after hepatitis C therapy: a prospective cohort study[END_REF][START_REF] Loko | The French national prospective cohort of patients co-infected with HIV and HCV (ANRS CO13 HEPAVIH): early findings, 2006-2010[END_REF][START_REF] Prentiss | Patterns of Marijuana Use among Patients with HIV/AIDS Followed in a Public Health Care Setting[END_REF]) and 13 years (IQR [START_REF] Chew | Short Communication: Coronary Heart Disease Risk by Framingham Risk Score in Hepatitis C and HIV/Hepatitis C-Coinfected Persons[END_REF][START_REF] Knight | Significant reductions in alcohol use after Hepatitis C treatment: Results from the French ANRS CO13-HEPAVIH cohort: Significant Reductions in Alcohol Use[END_REF][START_REF] Fialho | Depression in HIV and HCV co-infected patients: a systematic review and meta-analysis[END_REF][START_REF] Cacoub | Burden of HIV and hepatitis C co-infection: the changing epidemiology of hepatitis C in HIV-infected patients in France[END_REF][START_REF] Hagan | HIV/hepatitis C virus co-infection in drug users: risk behavior and prevention[END_REF][START_REF] Kariuki | HIV and smoking: associated risks and prevention strategies[END_REF][START_REF]Life expectancy of individuals on combination antiretroviral therapy in high-income countries: a collaborative analysis of 14 cohort studies[END_REF][START_REF] Morlat | Causes of death among HIV-infected patients in France in 2010 (national survey): trends since 2000[END_REF]), respectively. At baseline, 10.7% were HCV cured, while at the end-of-study visit, 19.6% were HCV cured.

Factors associated with TSQA

In univariable analyses (Table 1), the following explanatory variables were associated with TSQA: HCV cure, cannabis use (dichotomous variable (see above)), age, BMI, HIV transmission mode, and recent substance use.

The following variables were retained in the final multivariable model: HCV cure, BMI, HIV transmission mode, cannabis use and other illegal substance use.

The multivariable analysis confirmed the positive associations between HCV cure and TSQA (adjusted hazard ratio (HR) [95% CI]: 1.76 [1.06-2.93], p=0.029) (Figure 2), between a higher BMI and TSQA (1.06 [1.00-1.13], p=0.040), and between MSM transmission mode and TSQA (2.60 [1.44-4.70], p=0.002, versus IDU) (Table 1). By contrast, cannabis use (0.63 [0.40-1.00], p=0.049) (Figure 3) and recent illegal psychoactive substance use (0.35 [0.13-0.99], p=0.048) were independently negatively associated with TSQA. Older age was no longer significantly associated with the outcome in the multivariable analysis. The proportionality of hazards assumption was verified for all explanatory variables in the final multivariable Cox model (see Figure 2 for HCV cure status and Figure 3 for cannabis use).

Discussion

In this large cohort of HIV-HCV co-infected people in France, HCV cure was associated with more than a 75% higher chance of TSQA. Recent cannabis use was associated with a 37% lower chance of TSQA. Other illegal psychoactive substance use was also identified as a main barrier to TSQA.

To our knowledge, the association we found between HCV cure and TSQA has not been previously reported. However, it does reflect data from a large cohort of HCV-infected veterans in the USA, where those who were treated were less likely to be current smokers than the control group [START_REF] Butt | Direct-Acting Antiviral Therapy for HCV Infection Is Associated With a Reduced Risk of Cardiovascular Disease Events[END_REF]. Moreover, our results for TSQA reflect observations in HCV-infected IDU, for whom treatment appeared to have a positive impact on injecting behaviors, injection equipment sharing behaviors [START_REF] Caven | Impact of Hepatitis C treatment on behavioural change in relation to drug use in people who inject drugs: A systematic review[END_REF], and hazardous alcohol use [START_REF] Midgard | Changes in risk behaviours during and following treatment for hepatitis C virus infection among people who inject drugs: The ACTIVATE study[END_REF].

From a wider perspective, TSQA may be a manifestation of the psychosocial benefits of HCV cure. More specifically, it has been shown that HCV cure has a short-term positive impact on health-related quality of life, with noticeable improvements in anxiety/depression [START_REF] Juanbeltz | Impact of successful treatment with direct-acting antiviral agents on health-related quality of life in chronic hepatitis C patients[END_REF]. One could expect therefore that the disappearance of HCV-related stress [START_REF] Castera | Psychological impact of chronic hepatitis C: comparison with other stressful life events and chronic diseases[END_REF] alleviates the need to smoke in order to cope with stress [START_REF] Revell | Smoking as a coping strategy[END_REF][START_REF] Fidler | Self-perceived smoking motives and their correlates in a general population sample[END_REF] and therefore fosters TSQA. In people with a history of substance use, HCV treatment and/or HCV cure has been associated with increased self-care [START_REF] Batchelder | Hepatitis C treatment turned me around:" Psychological and Behavioral Transformation Related to Hepatitis C Treatment[END_REF][START_REF] Torrens | Beyond clinical outcomes: the social and healthcare system implications of hepatitis C treatment[END_REF], ability and motivation to plan for the future, self-confidence and empowerment [START_REF] Torrens | Beyond clinical outcomes: the social and healthcare system implications of hepatitis C treatment[END_REF][START_REF] Williams | Life projects: the transformative potential of direct-acting antiviral treatment for hepatitis C among people who inject drugs[END_REF]. These benefits are likely to foster TSQA. As smoking-related deaths become more common than liver-related deaths in HIV-HCV co-infected patients -thanks to HCV treatment scale-up (20) -HCV cure may participate in raising awareness of tobacco-related harms and promote TSQA. HCV cure may also be a proxy of social support [START_REF] Janda | The Effect of Psychosocial Factors on Success Rates of Hepatitis C Treatment[END_REF], which fosters TSQA [START_REF] De Dios | The Influence of Social Support on Smoking Cessation Treatment Adherence Among HIV+ Smokers[END_REF][START_REF] Soulakova | Motivational Benefits of Social Support and Behavioural Interventions for Smoking Cessation[END_REF]. Furthermore, it may also reflect better engagement in care [START_REF] Adeyemi | Hepatitis C treatment eligibility in an urban population with and without HIV coinfection[END_REF][START_REF] Dillon | Urgent action to fight hepatitis C in people who inject drugs in Europe[END_REF][START_REF] Dever | Engagement in Care of High-Risk Hepatitis C Patients with Interferon-Free Direct-Acting Antiviral Therapies[END_REF], implying more frequent exposure to and/or better accepted short interventions or counselling about tobacco use [START_REF] Socio | Is it feasible to impact on smoking habits in HIV-Infected patients? Mission impossible from the STOPSHIV Project cohort[END_REF][START_REF] Wray | A Meta-Analysis of Brief Tobacco Interventions for Use in Integrated Primary Care[END_REF].

All these results from the literature highlight the opportunity that medical visitswhether during or after HCV treatment -represent for screening, interventions and referral to a specialist smoking cessation service [START_REF] Socio | Is it feasible to impact on smoking habits in HIV-Infected patients? Mission impossible from the STOPSHIV Project cohort[END_REF][START_REF] Tesoriero | Smoking among HIV positive New Yorkers: prevalence, frequency, and opportunities for cessation[END_REF][START_REF] Bains | Patients' and healthcare professionals' views on a specialist smoking cessation service delivered in a United Kingdom hospital: a qualitative study[END_REF][START_REF] Khara | Smoking cessation outcomes of referral to a specialist hospital outpatient clinic[END_REF].

To our knowledge, this is also the first longitudinal study to show an association between cannabis use and TSQA in PLWH, in HCV mono-infected persons, and in HIV-HCV co-infected persons. Our results reflect those in other studies on PLWH which found associations between cannabis use and tobacco smoking status [START_REF] Benard | Tobacco addiction and HIV infection: toward the implementation of cessation programs. ANRS CO3 Aquitaine Cohort. AIDS Patient Care STDs[END_REF][START_REF] Edwards | Understanding the Prevalence of Smoking Among People Living with HIV (PLHIV) in Australia and Factors Associated with Smoking and Quitting[END_REF]. A lower likelihood of smoking cessation in cannabis users than in non-users has also been documented in the general population, both for nontreatment seeking (57) and treatment-seeking smokers [START_REF] Voci | Is cannabis use associated with tobacco cessation outcome? An observational cohort study in primary care[END_REF]. Larger studies are needed to confirm our primary result of the association between cannabis use and TSQA. However, given the negative impact of tobacco smoking on survival in HIV-HCV co-infected patients [START_REF] Santos | HCV-Related Mortality Among HIV/HCV Co-infected Patients: The Importance of Behaviors in the HCV Cure Era (ANRS CO13 HEPAVIH Cohort)[END_REF][START_REF] Klein | Mortality in HIV-hepatitis C co-infected patients in Canada compared to the general Canadian population (2003-2013)[END_REF], we strongly recommend that this result be taken into account immediately in clinical management and public health strategies.

Mechanisms fostering co-occurring tobacco and cannabis use have been identified, as have the potential benefits of dual abstinence [START_REF] Agrawal | The co-occurring use and misuse of cannabis and tobacco: a review[END_REF]. One possible major reason for cannabis use being a barrier to TSQA is that smoking is the primary route of cannabis administration. This is especially true in Europe [START_REF] Hindocha | No Smoke without Tobacco: A Global Overview of Cannabis and Tobacco Routes of Administration and Their Association with Intention to Quit[END_REF]. Other possible mechanisms include exposure to smoking cues (60) (which are maintained when tobacco is substituted with cannabis (61)), the putative potentiation of nicotine dependence by cannabis [START_REF] Dierker | Nicotine dependence predicts cannabis use disorder symptoms among adolescents and young adults[END_REF][START_REF] Cohen | SR141716, a central cannabinoid (CB(1)) receptor antagonist, blocks the motivational and dopamine-releasing effects of nicotine in rats[END_REF], and a desire to attenuate nicotine's undesirable effects or withdrawal symptoms [START_REF] Rabin | A review of co-morbid tobacco and cannabis use disorders: Possible mechanisms to explain high rates of co-use[END_REF]. There is also evidence that detrimental neurocognitive effects in long-term cannabis users (65) may foster persistent tobacco use [START_REF] Ashare | Optimizing treatments for nicotine dependence by increasing cognitive performance during withdrawal[END_REF]. Interestingly, we did not find any dose-dependent relationship between the frequency of cannabis use and tobacco TSQA.

Our results add evidence to the need to manage addictive behaviors using a holistic approach. It has been shown that having multiple concurrent substance use disorders is associated with a greater likelihood of substance use disorder persistence at a distance of three years (compared with having only one such disorder), and also with nicotine dependence [START_REF] Mccabe | The Three-Year Course of Multiple Substance Use Disorders in the United States: A National Longitudinal Study[END_REF]. The positive effects of smoking cessation on substance use outcomes have previously been highlighted [START_REF] Mckelvey | Impact of quitting smoking and smoking cessation treatment on substance use outcomes: An updated and narrative review[END_REF]. However, substance substitution mechanisms have also been reported [START_REF] Subbaraman | Substitution and Complementarity of Alcohol and Cannabis: A Review of the Literature[END_REF][START_REF] Allsop | Changes in cigarette and alcohol use during cannabis abstinence[END_REF].

Moreover, as some genetic vulnerabilities to addictions are related to pathways which are common to different substances (e.g., the reward pathway) (71), therapeutic programs targeting multiple substance use are likely to bring greater benefits than those only focusing on one substance. However, these programs would require treatment providers to undergo more in-depth training in order to be able to provide addiction care for a wide range of substances rather than just one. They would also require patients who are willing and motivated to simultaneously reduce or stop couse.

It is increasingly recognized that cannabis-and cannabinoid-based medicines provide relief for PLHW and HCV-infected persons [START_REF] Prentiss | Patterns of Marijuana Use among Patients with HIV/AIDS Followed in a Public Health Care Setting[END_REF][START_REF] Fischer | Treatment for hepatitis C virus and cannabis use in illicit drug user patients: implications and questions[END_REF][START_REF] Santos | HCV-Related Mortality Among HIV/HCV Co-infected Patients: The Importance of Behaviors in the HCV Cure Era (ANRS CO13 HEPAVIH Cohort)[END_REF]. In the general population, cannabis -whether used for recreational or medical purposes -is a barrier to tobacco cessation [START_REF] Voci | Is cannabis use associated with tobacco cessation outcome? An observational cohort study in primary care[END_REF]. Accordingly, improving access to medical cannabis and cannabinoids which are tobacco-free and which are not administered through smoking [START_REF] Loflin | No smoke, no fire: What the initial literature suggests regarding vapourized cannabis and respiratory risk[END_REF][START_REF] Lanz | Medicinal Cannabis: In Vitro Validation of Vaporizers for the Smoke-Free Inhalation of Cannabis[END_REF], would appear to be an urgent public health issue, especially for PLWH and HCV-infected people. Our findings may also encourage research on 'cannabis substitution' as a therapeutic support mechanism for HIV-infected, HCVinfected and HIV-HCV co-infected patients who attempt to stop smoking tobacco. To date, nabiximols have shown mixed results as a cannabinoid agonist treatment [START_REF] Trigo | Nabiximols combined with motivational enhancement/cognitive behavioral therapy for the treatment of cannabis dependence: A pilot randomized clinical trial[END_REF][START_REF] Lintzeris | Nabiximols for the Treatment of Cannabis Dependence: A Randomized Clinical Trial[END_REF][START_REF] Lintzeris | Cannabis use in patients 3 months after ceasing nabiximols for the treatment of cannabis dependence: Results from a placebo-controlled randomised trial[END_REF].

More generally, substance use, including alcohol, has been identified as a barrier to smoking cessation in HIV-infected populations [START_REF] Rahmanian | Cigarette Smoking in the HIV-Infected Population[END_REF]. Unsurprisingly therefore, in our study, recent use of psychoactive substances other than cannabis was associated with a 65% lower chance of TSQA. This reflects Encrenaz et al.'s finding of a lower TSQA incidence in IDU-infected PLWH [START_REF] Encrenaz | Determinants of smoking cessation attempts among HIV-infected patients results from a hospital-based prospective cohort[END_REF]. This finding may be partly due to greater baseline cigarette use and/or nicotine dependence (6), although we did not have these data in the present study. Furthermore, in line with Encrenaz et al. 's findings, the positive relationship we found between MSM HIV transmission mode and TSQA is likely due to the fact that our study population mainly comprised participants infected by HIV through IDU.

The positive association we found between BMI and TSQA is counter-intuitive.

As weight gain is a widely-known consequence of tobacco cessation, it may hinder motivation or confidence to stop tobacco smoking [START_REF] Weinberger | Perceived risks and benefits of quitting smoking in a sample of adults living with HIV/AIDS[END_REF][START_REF] Peretti-Watel | Smoking motivations and quitting motivations among HIV-infected smokers[END_REF]. Consequently, one would expect people with higher BMI to be less likely to try to quit. However, it is also possible that a higher BMI is a proxy for previous TSQA [START_REF] Chaiton | Estimating the number of quit attempts it takes to quit smoking successfully in a longitudinal cohort of smokers[END_REF].

Depression was not a barrier to TSQA in our study. This reflects Benard et al. who found no association between depressive symptoms and motivation to stop smoking in a French cohort of HIV-infected people (55). In the general population, depression appeared to be a barrier to quitting [START_REF] Shahab | Reciprocal associations between smoking cessation and depression in older smokers: findings from the English Longitudinal Study of Ageing[END_REF]. This absence of any association may be due to collinearity with cannabis use. Indeed, cannabis is frequently used by PLWH to cope with depression [START_REF] Harris | Patterns and correlates of cannabis use among individuals with HIV/AIDS in Maritime Canada[END_REF], and depression and cannabis use have been associated with each other in the general population [START_REF] Hasin | Cannabis Use, Cannabis Use Disorder, and Comorbid Psychiatric Illness: A Narrative Review[END_REF], as well as in PLWH [START_REF] Pacek | CANNABIS USE AMONG PERSONS LIVING WITH HIV IN THE UNITED STATES: FINDINGS FROM A NATIONALLY REPRESENTATIVE SAMPLE[END_REF].

Unlike Regan et al., who found that patients with a detectable HIV viral load were significantly less likely to quit smoking than their virologically suppressed counterparts (86), we did not find any association between HIV plasma load and our study outcome.

The large sample size and the relatively long follow-up duration are the main strengths of our study. However, several limitations must be acknowledged. First, unfortunately, our study sample selection did not lead to a study sample representative of the smokers in the original cohort (which is itself representative of HIV-HCV co-infected people engaged in care in French hospitals), and duplication of our results would be needed to ensure their generalizability. Second, due to the interval-censored nature of the smoking status data collected during follow-up in the cohort, we may have missed short-lived TSQA which occurred between two annual or biannual (for people with cirrhosis) follow-up visits. Third, the LOCF method used to account for missing data on explanatory variables is grounded in the assumption of stability of these variables over time. As variables were mainly socio-behavioral, we expect this assumption to be true, although we cannot confirm it. Fourth, we were not able to characterize the duration of tobacco use or abstinence, and did not have information on whether TSQA were self-helped or assisted. Finally, many patients did not provide information on the number of cigarettes they smoked, and therefore data on this variable could not be used here. Moreover, we had no information about the factors related to people's motivation to quit smoking. However, as TSQA may be considered part of the process leading to quitting [START_REF] Chaiton | Estimating the number of quit attempts it takes to quit smoking successfully in a longitudinal cohort of smokers[END_REF], identifying modifiable factors which facilitate them is of great clinical importance. Along with modifiable factors, future research should explore the possible environmental factors (e.g., the presence of smokers in the household) which may facilitate or impede TSQA.

To conclude, HCV cure was associated with a higher chance of tobacco smoking quit attempts in the present study of HIV-HCV co-infected patients, a population characterized by a high prevalence of both tobacco smoking and cannabis use, and which is especially vulnerable to tobacco-related harms and mortality.

Conversely, cannabis use was associated with a lower chance of quit attempts.

Tobacco smoking should be systematically addressed during standard care of HIV-HCV infected people. HCV treatment represents a good opportunity for screening, comprehensive counselling, and referral to specialist tobacco cessation services.

Cannabis use should be screened concomitantly with tobacco use in HIV-infected, HCV-infected and HIV-HCV co-infected smokers. Finally, for cannabis users who wish to quit smoking tobacco, safer routes of cannabis administration should be promoted which do not involve smoking cues such as vaporization. This would reduce associated harms and help users in future tobacco smoking cessation attempts. * Descriptive statistics of time-dependent variables are given for the lagged variables (measured at n-1). 1 Employed, retired, being a housewife/househusband. 2 Cannabis use was entered as a dichotomous variable in the final multivariable model as the alternate three-level variable was no longer significantly associated with the outcome during the stepwise procedure. 3 Illegal psychoactive substance use (excluding cannabis) in the previous 4 weeks. The illegal psychoactive substances evaluated were cocaine, heroin, crack, ecstasy, street Subutex, amphetamines and LSD/other hallucinogens. 4 Advanced liver fibrosis was defined as an FIB-4 index >3.25.

CD4 count (in 100 cells/mm

P

  . Morlat, F. Bani-Sadr, L. Meyer, F. Boufassa, , B. Autran, A.M. Roque, C. Solas, H. Fontaine, D. Costagliola, L. Piroth, A. Simon, D. Zucman, F. Boué, P. Miailhes, E. Billaud, H. Aumaître, D. Rey, G. Peytavin, V. Petrov-Sanchez, A. Levier. Clinical Centers (ward / participating physicians): APHP, Hôpitaux Universitaires Paris Centre, Paris (Médecine Interne et Maladies Infectieuses : D. Salmon, R. Usubillaga; Hépatogastro-entérologie : P. Sogni ; Anatomo-pathologie : B. Terris ; Virologie :P. Tremeaux) ; APHP Pitié-Salpétrière, Paris (Maladies Infectieuses et Tropicales : C. Katlama, M.A. Valantin, H. Stitou ; Médecine Interne : A. Simon, P. Cacoub, S. Nafissa ; Hépato-gastro-entérologie : Y. Benhamou ; Anatomo-pathologie : F. Charlotte ; Virologie : S. Fourati) ; APHM Sainte-Marguerite, Marseille (Service d'Immuno-Hématologie Clinique : I. Poizot-Martin, O. Zaegel, H. Laroche ; Virologie : C. Tamalet) ; APHP Tenon, Paris (Maladies Infectieuses et Tropicales : G. Pialoux, J. Chas; Anatomo-pathologie : P. Callard, F. Bendjaballah ; Virologie : C. Amiel, C. Le Pendeven) ; CHU Purpan, Toulouse (Maladies Infectieuses et Tropicales : B. Marchou ; Médecine interne : L. Alric ; Hépato-gastro-entérologie : K. Barange, S. Metivier ; Anatomopathologie : J. Selves ; Virologie : F. Larroquette) ; CHU Archet, Nice (Médecine Interne : E. Rosenthal ; Infectiologie : A. Naqvi, V. Rio ; Anatomo-pathologie : J. Haudebourg, M.C. Saint-Paul ; Virologie : A. De Monte, V. Giordanengo, C. Partouche) ; APHP Avicenne, Bobigny (Médecine Interne -Unité VIH : O. Bouchaud ; Anatomo-pathologie : A. Martin, M. Ziol ; Virologie : Y. Baazia, V. Iwaka-Bande, A. Gerber) ; Hôpital Joseph Ducuing, Toulouse (Médecine Interne : M. Uzan, A. Bicart-See, D. Garipuy, M.J. Ferro-Collados ; Anatomopathologie : J. Selves ; Virologie : F. Nicot) ; APHP Bichat -Claude-Bernard, Paris (Maladies Infectieuses :, A. Gervais, Y. Yazdanpanah; Anatomo-pathologie : H. Adle-Biassette ; Virologie : G. Alexandre, Pharmacologie : G. Peytavin) ; APHP Saint-Louis, Paris (Maladies infectieuses : C. Lascoux-Combe, J.M. Molina; Anatomo-pathologie : P. Bertheau; Virologie :M.L. Chaix, C. Delaugerre, S. Maylin) ; APHP Saint-Antoine (Maladies Infectieuses et Tropicales : K. Lacombe, J. Bottero; J. Krause, P.M. Girard, Anatomo-pathologie : D. Wendum, P. Cervera, J. Adam ; Virologie : C. Viala) ; APHP, Hôpitaux Paris Sud, Bicêtre, Paris (Maladies Infectieuses et Tropicales : D. Vittecocq ; Médecine Interne : C. Goujard, Y. Quertainmont, E. Teicher; Virologie : C. Pallier) ; APHP Necker, Paris (Maladies Infectieuses et Tropicales : O. Lortholary, C. Duvivier, C. Rouzaud, J. Lourenco, F. Touam, C. Louisin : Virologie : V. Avettand-Fenoel, E. Gardiennet, A. Mélard) ; CHU Bordeaux Hôpital Pellegrin, Bordeaux (Maladies Infectieuses et Tropicales : D. Neau, A. Ochoa, E. Blanchard, S. Castet-Lafarie, C. Cazanave, D. Malvy, M. Dupon, H. Dutronc, F. Dauchy, L. Lacaze-Buzy, A. Desclaux; Anatomo-pathologie : P. Bioulac-Sage ; Virologie : P. Trimoulet, S. Reigadas) ; CHU Bordeaux Hôpital Saint-André, Bordeaux (Médecine Interne et Maladies Infectieuses : P. Morlat, D. Lacoste, F. Bonnet, N. Bernard, M. Hessamfar, J, F. Paccalin, C. Martell, M. C. Pertusa, M. Vandenhende, P. Mercié, D. Malvy, T. Pistone, M.C. Receveur, M. Méchain, P. Duffau, C Rivoisy, I. Faure, S. Caldato ; Anatomo-pathologie : P. Bioulac-Sage ; Virologie : P. Trimoulet, S. Reigadas, P. Bellecave, C. Tumiotto) ; CHU Bordeaux Hôpital du Haut-Levêque, Bordeaux (Médecine Interne : J.L. Pellegrin, J.F. Viallard, E. Lazzaro, C. Greib ; Anatomopathologie : P. Bioulac-Sage ; Virologie : P. Trimoulet, S. Reigadas) ; Hôpital FOCH, Suresnes (Médecine Interne : D. Zucman, C. Majerholc ; Virologie : M. Brollo, E. Farfour) ; APHP Antoine Béclère, Clamart (Médecine Interne : F. Boué, J. Polo Devoto, I. Kansau, V. Chambrin, C. Pignon, L. Berroukeche, R. Fior, V. Martinez, S. Abgrall, M. Favier ; Virologie : C. Deback) ; CHU Henri Mondor, Créteil (Immunologie Clinique : Y. Lévy, S. Dominguez, J.D. Lelièvre, A.S. Lascaux, G. Melica) ; CHU Nantes Hôpital Hôtel Dieu, Nantes (Maladies Infectieuses et Tropicales : E. Billaud, F. Raffi, C. Allavena , V. Reliquet, D. Boutoille, C. Biron; M. Lefebvre, N. Hall, S. Bouchez ; Virologie : A. Rodallec, L. Le Guen, C. Hemon) ; Hôpital de la Croix Rousse, Lyon (Maladies Infectieuses et Tropicales : P. Miailhes, D. Peyramond, C. Chidiac, F. Ader, F. Biron, A. Boibieux, L. Cotte, T. Ferry, T. Perpoint, J. Koffi, F. Zoulim, F. Bailly, P.

  [kg] divided by height squared [m 2 ].

Figure 3 :

 3 Figure 3: Probability of no tobacco smoking cessation attempt during follow-up according to cannabis use.

Figure 1

 1 Figure 1

3 )(n=694)*

  Descriptive statistics are given for the end-of-study visit. Median and interquartile range are given for continuous variables.

		5.3 [3.4-			0.94	0.88-1.02	0.143
		7.4]
	Undetectable plasma HIV
	viral load (n=691)*
	No (ref.)	162	23.4	27	1
	Yes	529	76.6	60	0.73	0.46-1.17	0.190
	Time since HCV	13 [10-			1.01	0.97-1.05	0.707
	diagnosis (in years)	17]
	(n=680)*
	HCV cure*
	No (ref.)	559	80.4	66	1			1
	Yes	136	19.6	22	1.94	1.18-3.18	0.009	1.76	1.06-2.93	0.029
	Advanced liver fibrosis 4 *
	No (ref.)	569	81.9	74	1
	Yes	126	18.1	14	1.08	0.60-1.92	0.806
	Depressive symptoms
	(n=660)*
	No (ref.)	394	59.7	58	1
	Yes	266	40.3	28	0.68	0.43-1.08	0.101
	History of treatment for
	depression 5 (n=694)*
	No (ref.)	354	51.0	54	1
	Yes	340	49.0	34	0.70	0.45-1.08	0.109
	Total number of self-	11 [5-			1.00	0.97-1.03	0.962
	reported symptoms 6	18]
	(n=674)*

#

This study was sponsored and funded by the French National Agency for Research on Aids and Viral Hepatitis (ANRS).

This study was sponsored and funded by the French National Agency for Research on Aids and Viral Hepatitis (ANRS). The authors thank all the members of the ANRS-CO-13-HEPAVIH study group. Special thanks to all the physicians and nurses involved in the follow-up of the cohort and all patients who took part in this study. Finally, we thank Jude Sweeney for the English revision and editing of our manuscript. . Salmon (co-Principal investigator), L.

no data are available.

Code availability: not applicable.

Ethics approval: The study was designed and implemented in accordance with the Declaration of Helsinki, and the protocol was approved by the ethics Committee of the Cochin University Hospital in Paris.

Consent to participate: all participants provided written informed consent.


Camille Cabrol
email: camcabrol@gmail.com

Léa Elarouti
email: lea.elarouti@outlook.fr

Anne-Laure Montava
email: anne-laure.montava@wanadoo.fr

Sylvie Jarze
email: sylvie.jarze@icloud.com

Julien Mancini
email: julien.mancini@ap-hm.fr

Jean-Pierre Lavieille
email: jean-pierre.lavieille@ap-hm.fr

Pauline Barry
email: pauline.barry@ap-hm.fr

Marion Montava
email: marion.montava@ap-hm.fr

Elarouti Léa

Jarze Sylvie

Lavieille Jean-Pierre

Barry Pauline

M D Montava Marion

Sunnybrook Facial Grading System: Intra-rater and Inter-rater Variabilities

Keywords: Facial palsy, Facial grading scales, Sunnybrook Facial Grading System, Inter-rater variability, Intra-rater variability

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Sunnybrook Facial Grading System: Intra-rater and inter-rater variabilities ABSTRACT:

Objectives: Evaluate intra-rater and inter-rater variabilities of the Sunnybrook Facial Grading System (SFGS) and identify potential factors of variability.

Study design: Prospective test of hypothesis.

Setting: University tertiary referral centre.

Participants/Methods: Facial video recordings of 20 patients with variable degrees of peripheral facial palsy (PFP) were anonymized then randomly presented to 31 independents raters in two trials. The raters were senior and junior professionals involved in the management of PFP: ENT specialists, physiotherapists and speech therapists. The SFGS was used for grading paralyses.

Main outcome measure: Intra-rater and inter-rater variabilities were estimated by intraclass correlation coefficient (ICC [95% confidence interval]) for the composite score and the three subscores of the SFGS. Factors of variability studied were: rater professions and rater experience (senior vs junior).

Results: For the total population, the intra-rater ICC was 0.915 [0.900-0.929] for the composite score considered to represent almost perfect repeatability. Repeatability was important for symmetry at rest (0.694 [0.646-0.737]), almost perfect for voluntary movements (0.903 [0.886-0.918]) and synkinesis (0.810 [0.778-0.838]). The inter-rater ICC for the composite score was 0.847 [0.755-0.923] indicating almost perfect agreement between all raters. Agreement between raters was almost perfect for voluntary movements (0.839 [0.746-0.919]), but moderate for symmetry at rest (0.553 [0.408-0.730]) and synkinesis (0.476 [0.333-0.666]). Some differences were found between raters groups; however, repeatability and agreement were good for all raters.

INTRODUCTION

Peripheral facial palsy (PFP) is a pathology caused by a facial nerve injury which affects the motricity of the hemiface on the side of the lesion. Clinicians need an objective, reliable and reproducible clinical tool to accurately describe motor facial function, assess the severity of paralysis, its evolution over time and the effects of treatments.

To this day, a multitude of different facial nerve grading instruments were developed which shows that none of them is perfect and how difficult it is to assess PFP. In 2015, a systematic review on the facial function grading instruments found that the Sunnybrook Facial Grading System (SFGS) was the scale which best accomplishes the goals of assessment and recommended its widespread adoption as the current standard in reporting outcomes of facial nerve disorders [START_REF] Fattah | Facial Nerve Grading Instruments: Systematic Review of the Literature and Suggestion for Uniformity[END_REF]. Indeed, the House and Brackmann Grading system is the best known and most widely used system for its general ease of use. However, it is not giving specific details about facial function.

Introduced by Ross in 1996 (2), SFGS assesses facial resting symmetry compared to normal side, symmetry of voluntary movement and potential synkinesis associated with specified voluntary movement. Three subscores are obtained giving a composite score from 0 for complete PFP to 100 for normal facial function. However, the SFGS remains a subjective scale which makes it subject to limitation because of varying degrees of variability. From 2000 to 2010, some studies on SFGS's variability concluded that SFGS was generally reliable (3-7).

Nevertheless, they found contradictory results of variability for individual scores.

The main objective of our study was to evaluate intra-rater and inter-rater variabilities of the SFGS. Secondly, we tried to identify potential factors of variability as the profession or the level of experience in the management of patients with PFP.

MATERIALS AND METHODS

Ethical considerations

The study protocol was assessed according to guidelines of the national committee on research involving human subjects and was approved by Institutional Review Board (Ethics committee of Aix Marseille University 2019-17-10-001).

Study protocol

Subject assembly was possible through access to the facial nerve centre perpetual database comported video archive of subjects with PFP. This resource is under the control of the last author and protected behind a two-lock secure system.

After statistical consultation and review of literature, it was arbitrarily determined that 20 subjects would be sufficient to test the variability of the scale. The inclusion criteria were: goodquality video recording, unilateral PFP, all five facial voluntary expressions necessary for SFGS scoring present, and a wide range and gradated distribution of facial movement among the 20 subjects' video images. Subjects with facial trauma were excluded. Diagnosis, time course in recovery, age, gender, or race were not considered criteria for selection, and the videotapes were anonymized. The selection of videotapes and the randomised order of viewing were determined by a committee of three experts.

A group of 31 independent raters was constituted by senior and junior professionals involved in the management of patients with PFP: ENT specialists, physiotherapists and speech therapists. The raters analysed the videotapes and graded the facial function of each subject using the SFGS and the HBGS in 2 independent and timed rounds with a 2-months interval.

Neither documentation nor training session were provided. During session, the raters could pause or go back at any time if needed to have a better analysis as long as the chronometer was not stopped. The time of evaluation was reported. Moreover, information about their experience in the management of patients with PFP and a signed consent were reported.

Statistical analysis

To estimate intra-rater and inter-rater variabilities, the intraclass correlation coefficient (ICC [95% confidence interval]) was estimated [START_REF] Deyo | Reproducibility and responsiveness of health status measures. Statistics and strategies for evaluation[END_REF]. ICC was estimated for the composite score, the three subscores and each movement for the total population and for each group of raters.

According to Landis Koch (Biometrics, 1977), we considered the agreement as weak if rated within 0-0.40, moderate within 0.41-0.60, important within 0.61-0.80 and almost perfect within 0.81-0.99. Analysis were carried out with SPSS 20.0 for Windows.

Inter-rater variability was studied in the first session in order to avoid a learning effect even if the 2-months interval should be sufficient for this.

As ICC can reflect the degree of agreement but also relate to the number and the variability among the sampled observations, we did not compare statistically different measurements or raters. However, if the confidence intervals of the ICC were different without overlap between two groups, the reliability was considered to be different, better or worse.

RESULTS

In the first session, 31 raters participated: 6 ENT specialists (4 seniors and 2 juniors), 4 physiotherapists (3 seniors and 1 junior) and 21 speech therapists (9 seniors and 12 juniors). In the second session, 25 of them (80.6%) took part: 5 ENT specialists (3 seniors and 2 juniors), 3 physiotherapists (2 seniors and 1 junior) and 17 speech therapists (6 seniors and 11 juniors).

The average time of evaluation was 64.9 minutes (min) for both sessions with a standard deviation of 22.8. It was 70.9 ± 23.4 min for session 1, and 57.5 ± 19.8 min for session 2.

The information gathering highlighted the following points: all juniors had had a former PFP teaching, however, only 3/15 (20%) of them had already used the SFGS; and 12/16 (75%) of senior had at least one training course on PFP but only 9 (56.3%) used SFGS in their clinical practice.

Intra-rater variability (repeatability)

Results of intra-rater variability for the composite score and the three subscores are reported in Results of intra-rater variability for the five standard expressions in voluntary movement and synkinesis are reported Table 2. For voluntary movement, agreement was almost perfect for the five movements performed. For synkinesis, repeatability was moderate for the gentle eye closure (0.600 [0.541-0.653]) while for the four other movements it was important.

No difference of repeatability was found between our raters groups of seniors and juniors, except for resting symmetry with better repeatability for seniors (0.791 [0.736-0.836]) than for juniors (0.620 [0.543-0.687]) and synkinesis with again better repeatability for seniors (0.880 [0.847-0.907]) than for juniors (0.726 [0.665-0.777]).

No difference was found between our raters groups of professions, except for the composite score and voluntary movement with better repeatability for ENT specialists and speech therapists than for physiotherapists.

Inter-rater variability (agreement between raters)

Results of inter-rater variability for the composite score and the three subscores are reported in No differences between seniors and juniors raters groups were reported. No difference was found between our raters groups of professions, except for voluntary movement where physiotherapists had worse agreement than ENT specialists and speech therapists.

For extreme HB grades (II and VI), agreement was almost perfect for the composite score (0.948 [0.873-0.991]) and the voluntary movement (0.954 [0.887-0.992]), important for resting symmetry (0.738 [0.511-0.945]) and weak for synkinesis (0.018 [-0.004-0.169]). For medium HB grades (III, IV, V), agreement was important for the composite score (0.691 [0.529-0.855]) and the voluntary movements (0.704 [0.546-0.863]), moderate for resting symmetry (0.461 [0.299-0.696]) and for synkinesis (0.427 [0.269-0.666]). Repeatability of extreme HB grades was better than medium HB grades for the composite score and voluntary movement, and worse for synkinesis. There was no difference for resting symmetry.

DISCUSSION

SFGS assesses resting symmetry compared to normal side, symmetry of voluntary movement and potential synkinesis associated with specified voluntary movement. Resting symmetry is assessed by a comparison to the normal side of the palpebral fissure (normal, narrow, wide), the naso-labial fold (normal, absent, less or more pronounced), and the corner of the mouth (normal, drooped or pulled up/out). The rating is done through a points-giving system (0, 1, 2).

Then, different regions of the face are examined separately, with five standard expressions used to assess the symmetry of voluntary movement and the degree of synkinesis associated with movement. The five standard expressions reflect the motor function of the five peripheral branches of the facial nerve: forehead wrinkle (frontalis), gentle eye closure (orbicularis oculi), open mouth smile (zygomaticus and risorius), snarl (levator labii superioris alaeque nasi and levator labii superioris) and lip pucker (orbicularis oris superior and inferior). The symmetry of voluntary movement for each standard expression is graded on a five-points scale from 1 (no movement) to 5 (movement complete), depending on the degree of muscle excursion compared to normal side. The degree of synkinesis associated with each standard expression is rated on a four-points scale from 0 (no synkinesis) to 3 (severe synkinesis). Three scores are obtained and weighted as follows: the resting symmetry score is multiplied by five, and the voluntary movement score is multiplied by four. Then, a composite score is calculated by subtracting the resting symmetry score, and the synkinesis score from the voluntary movement score. A composite score of 100 corresponds to normal facial function and a composite score of 0 to complete PFP.

As it is a scale administered by rater with their own clinical experience, the SFGS remains a subjective tool subject to limitation. Previous studies on SFGS's variability had some similar and contradictory outcomes [START_REF] Kayhan | Toronto Facial Grading System: Interobserver reliability[END_REF][START_REF] Hu | Reliability of the Sunnybrook Facial Grading System by novice users[END_REF][START_REF] Coulson | Reliability of the "Sydney," "Sunnybrook," and "House Brackmann" Facial Grading Systems to Assess Voluntary Movement and Synkinesis after Facial Nerve Paralysis[END_REF][START_REF] Kanerva | Sunnybrook and House-Brackmann Facial Grading Systems: Intrarater repeatability and interrater agreement[END_REF][START_REF] Neely | Sunnybrook facial grading system: Reliability and criteria for grading[END_REF]. Our purpose was to evaluate intra-and inter-rater variabilities of the SFGS in a new study with more patients and more raters. To go further, we decided to include raters from different professions and experiences. The statistical method chosen to evaluate intra-rater and inter-rater variabilities can have a marked effect on study outcome. The choice of ICC was coherent with those previous studies and then allowed a comparison of outcomes.

Intra-rater variability (repeatability)

In our study with 31 raters, repeatability evaluated by ICC for the composite score was 0.900 to 0.929. ICC varied from 0.838 to 0.929 with eight novice assessors in Hu et al. ( 4), 0.864 to 0.995 with 26 doctors in Kanerva et al. [START_REF] Kanerva | Sunnybrook and House-Brackmann Facial Grading Systems: Intrarater repeatability and interrater agreement[END_REF], 0.948 to 0.970 for 2 naïve raters in Neely et al. [START_REF] Neely | Sunnybrook facial grading system: Reliability and criteria for grading[END_REF]. No difference was found between our raters groups of professions, except for composite score and voluntary movement with better reproducibility for ENT specialists and speech therapists than for physiotherapists. It suggests that SFGS repeatability is the same for all professionals involved in the management of PFP, except for physiotherapists. This outcome has to be treated cautiously owing to the heterogeneous sizes of the raters groups included in our study.

Thus, the results of the intra-rater variability enable to conclude that the SFGS is a reproducible scale from one time to another.

Inter-rater variability (agreement between raters)

In our study with 31 raters, agreement evaluated by ICC for the composite score was 0.847 In our study, agreement was weak for forehead wrinkle, gentle eye closure, open mouth smile and snarl, and moderate for lip pucker. These results are opposite to Kayhan et al. who found an important agreement for each movement except frontal synkinesis which showed a weak agreement (0.38 [0.21-0.59]). Thus, agreement between raters for synkinesis was highly variable. Those discrepancies can be due to the fact that the rater has to observe the whole face, whereas for voluntary movement the rater's attention is directed to a specific region of the face.

It should be noted that in some previous studies [START_REF] Kayhan | Toronto Facial Grading System: Interobserver reliability[END_REF][START_REF] Hu | Reliability of the Sunnybrook Facial Grading System by novice users[END_REF][START_REF] Kanerva | Sunnybrook and House-Brackmann Facial Grading Systems: Intrarater repeatability and interrater agreement[END_REF][START_REF] Neely | Sunnybrook facial grading system: Reliability and criteria for grading[END_REF], raters had preliminary training before the first session which may have helped to reach higher results of agreement with SFGS than ours.

Differences between seniors and juniors raters groups were not significant. This result is consistent with Hu et al. and Kanerva et al. and suggest that the SFGS can be used with little prior knowledge of the scale and then as reliably by both experts and novice users.

No difference was found between our raters groups of professions, except for voluntary movement with better repeatability for ENT specialists and speech therapists than for physiotherapists. It suggests that agreement on SFGS is the same for all professionals involved in the management of PFP, except for physiotherapists. Again, this outcome has to be treated cautiously owing to the heterogeneous sizes of the raters groups.

Thus, the results of the inter-rater variability enable to conclude that the SFGS is a reproducible scale from one rater to another. To reduce some ambiguities due to subjective assessment, Neely and. al suggested criteria for completion of the SFGS.

Time of scoring

We reported the unequal times of scoring between session 1 (70.9 min) and session 2 (57.5 min). Knowing that a few raters use the SFGS in their practice (20% of juniors, 56.3% of seniors), we could suggest a more efficient using of the scale during session 2. It wouldn't be likely that there was a learning effect on patients as we set a 2-months interval between the two sessions to avoid this bias. This outcome has to be treated cautiously owing to the lost raters in the second session.

Limits of our study

Using videos could be considered as a limit as it is not as faithful as face to face assessment and adds doubts. Indeed, face to face examination offers the opportunity to ask the patient to repeat certain movements and may make the scoring easier.

Given that we are in close relationship with speech language therapists, their overrepresentation is due to a recruitment bias. This could reduce the power of our study to identify variability factors.

The raters inclusion was based on volunteering. The length of visioning and rating (64.89 min) explains the lost to follow up raters in the second session. It also brings to light the fact that, in clinical practice, the use of SFGS with all the subscores calculation is not practicable in emergency.

The comparison with the other studies was difficult as long as the methodologies were all different.

CONCLUSION

Based on our results, the SFGS is a reproducible scale from one time to another, showing an important too almost perfect repeatability. It is also reproducible from one rater to another, with almost perfect agreement for the composite score and voluntary movement, and moderate for resting symmetry and synkinesis.

The SFGS can be used with good reproducibility by both novices and experts, and by all professionals involved in the management of PFP.

DISCLOSURE

The authors report no conflict of interest.

  For extreme HB grades (II and VI), repeatability was almost perfect for the composite score (0.962 [0.947-0.972]) and the voluntary movement (0.961 [0.946-0.972]), important for resting symmetry (0.723 [0.636-0.792]) and moderate for synkinesis (0.576 [0.458-0.674]). For medium HB grades (III, IV, V), repeatability was almost perfect for the composite score (0.836 [0.802-0.865]) and the voluntary movement (0.828 [0.792-0.859]) and important for resting symmetry (0.677 [0.616-0.730]) and for synkinesis (0.786 [0.742-0.823]). Repeatability was better for the composite score and voluntary movement of extreme HB grades than medium HB grades. It was worse for synkinesis and there was no difference for resting symmetry.

  These results are considered to represent almost perfect repeatability.Although the repeatability score for resting symmetry was considered as important (0.694 [0.646-0.737]) in the current study, it was almost perfect for Kanerva et al. (0.841 [0.500-0.976]). However, Kanerva et al. specified that results with coefficient of repeatability (CR) varied for resting symmetry: repeatability was only moderate or fair. We found an almost perfect repeatability for the voluntary movement score (0.903 [0.886-0.918]) and for each movement performed. This result is consistent with the findings of Kanerva et al. (0.918 [0.799-0.988]). In our study, repeatability for the synkinesis was almost perfect (0.810 [0.778-0.838]). This result approaches the good findings of Kanerva et al. (0.979 [0.931-0.998]). No difference of repeatability was found between our raters groups of seniors and juniors for the composite score and voluntary movement. This result is congruent with Hu et al. and Kanerva et al. SFGS can be used as reliably by both experts and novice users. However, contrary to Hu et al. and Kanerva et al., resting symmetry and synkinesis shown better repeatability for seniors than for juniors which suggests that resting symmetry and synkinesis evaluation requires experience.

[ 0 .

 0 755-0.923]. It was 0.885 [0.76-0.92] in Kayhan et al. (3) with 5 ENT specialist, 0.892 in Hu et al. with 2 novice users, 0.997 [0.992-1.000] in Kanerva et al. with 26 doctors, 0.890 [0.784-0.946] in Neely et al. with 2 naïve raters. These results represent an almost perfect agreement between raters. We found a moderate agreement for symmetry at rest (0.563 [0.408-0.730]). However, Hu et al. found an almost perfect agreement (0.950) as Kanerva et al. (0.983 [0.960-0.996]). Kayhan et al. found an important agreement (0.72 [0.58-0.84]). In the current study, agreement between raters was almost perfect for voluntary movement (0.839 [0.746-0.919]). This outcome is congruent with Kayhan et al. (0.83 [0.73-0.90]), Hu et al. (0.976) and Kanerva et al. (0.997 [0.992-0.999]). Coulson et al. (5), with 6 ENT specialists, found important agreement (0.63). Agreement was almost found perfect for forehead wrinkle and open mouth smile, and important for gentle eye closure, snarl and lip pucker. Neely et al. specified that forehead wrinkle and lip pucker were most variable and significantly different from the other movements. For synkinesis we found moderated agreement (0.476 [0.333-0.666]). It was almost perfect in Hu et al. (0.913) and Kanerva et al. (0.987 [0.969-0.997]), but they didn't find as good results using CR to assess agreement: it was, only moderate or fair. Agreement was important for Kayhan et al. (0.70 [0.55-0.93]). Coulson et al. found a weak agreement (0.23).

Table 1 .

 1 The composite score ICC for the total population was 0.915 [0.900-0.929] considered to represent almost perfect repeatability. Repeatability was important for symmetry at rest

	(0.694 [0.646-0.737]), almost perfect for voluntary movement (0.903 [0.886-0.918]) and
	synkinesis (0.810 [0.778-0.838]).

Table 3 .

 3 The ICC for the composite score was 0.847 [0.755-0.923] indicating almost perfect agreement between all raters. Agreement was almost perfect for voluntary movement (0.839

	[0.746-0.919]), but moderate for symmetry at rest (0.553 [0.408-0.730]) and synkinesis (0.476
	[0.333-0.666]).
	Results of inter-rater variability for the five standard expressions in voluntary movement and
	synkinesis are reported Table 2. For voluntary movement, agreement was almost perfect for
	forehead wrinkle (0.805 [0.701-0.899]) and open mouth smile (0.816 [0.714-0.905]). It was
	important for gentle eye closure (0.724 [0.588-0.852]), snarl (0.754 [0.629-0.870]) and lip
	pucker (0.611 [0.467-0.774]). For synkinesis, agreement was weak for forehead wrinkle (0.387
	[0.258-0.582]), gentle eye closure (0.300 [0.188-0.488]), open mouth smile (0.373 [0.246-
	0.568]) and snarl (0.340 [0.219-0.532]). It was moderated for lip pucker (0.439 [0.302-0.632]).