Dr Antoine Yrondi
email: antoineyrondi@gmail.com

Federico Nemmi

Sophie Billoux

Aurélie Giron

Marie Sporer

Simon Taib

Juliette Salles

Damien Pierre

Claire Thalamas

Emilie Rigal

; Salles

; Rigal

; Pariente

; Schmitt

; Arbus

; Peran

Grey Matter changes in treatment-resistant depression during electroconvulsive therapy

Keywords: Treatment Resistant Depression, Electroconvulsive therapy, MRI, Cortical

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

Introduction 20-30% of depressed patients experience Treatment Resistant Depression (TRD).

Electroconvulsive Therapy (ECT) remains the treatment of choice for TRD. However, the exact mechanism of ECT remains unclear. We aim to assess grey matter changes in patients with TRD undergoing bilateral ECT treatment at different points during and after treatment.

Methods

Patients are recruited at the University Hospital of Toulouse. Eligibility criteria include a diagnosis of TRD and an age between 50 and 70 years old. Patients received clinical assessments (Hamilton Depression Rating Scale) and structural scans (MRI) at three points: baseline (within 48 h before the first ECT); V2 (after the first ECT considered effective); and thickness; Hippocampus; Amygdala.

Introduction

With more than 300 million people affected and a high lifetime prevalence of between 16-17% in the general population [START_REF] Kessler | Lifetime prevalence and age-of-onset distributions of DSM-IV disorders in the National Comorbidity Survey Replication[END_REF], major depressive disorder (MDD) is an increasingly widespread illness and a leading cause of disability worldwide (" WHO | Depression," n.d.). Beyond its social costs and economic burden [START_REF] Mrazek | A review of the clinical, economic, and societal burden of treatment-resistant depression: 1996-2013[END_REF], MDD induces both psychological and physical suffering through a broad range of health consequences [START_REF] Lépine | The increasing burden of depression[END_REF] (i.e. increased risks of suicidal behaviour or cardiovascular death). Treatment-resistant depression (TRD) has been estimated to represent half of the overall treatment costs for major depression [START_REF] Greden | The burden of disease for treatment-resistant depression[END_REF][START_REF] Olsen | Prevalence of major depression and stress indicators in the Danish general population[END_REF]. It can be assumed that approximately between 20-30% of depressed patients experience TRD [START_REF] Rush | Acute and longer-term outcomes in depressed outpatients requiring one or several treatment steps: a STAR*D report[END_REF], and up to one-half of patients only respond to treatment partially [START_REF] Fava | Definition and epidemiology of treatment-resistant depression[END_REF]. TRD is currently defined as the failure of at least two sequential courses of antidepressant treatments, administered at an appropriate dose and for the corresponding duration (Berlim and Turecki, 2007;[START_REF] Fava | Definition and epidemiology of treatment-resistant depression[END_REF]. TRD is known to have a heterogeneous aetiology. Some sources of variability include different environmental risk factors (e.g., childhood adversities), multiple genetic determinants (related to numerous genetic loci), and various epigenetic contributors (e.g., hypothalamic-pituitary-adrenal axis, immune function, monoamines, neurotrophic factors, etc. [START_REF] El-Hage | Mechanisms of antidepressant resistance[END_REF][START_REF] Fava | Definition and epidemiology of treatment-resistant depression[END_REF][START_REF] Greden | The burden of disease for treatment-resistant depression[END_REF][START_REF] Malhi | Treatmentresistant depression: resistant to definition?[END_REF][START_REF] Olsen | Prevalence of major depression and stress indicators in the Danish general population[END_REF]). Moreover, our clinical approach to TRD may be suboptimal. Despite these sources of variability, Electroconvulsive Therapy (ECT) continues to be the treatment of choice for severe TRD [START_REF] Fink | Electroconvulsive therapy: evidence and challenges[END_REF].

However, the exact mechanism of ECT remains unclear. There are many hypotheses that can explain its efficacy, albeit only in part: involvement of (i) monoamine pathway (for details, see [START_REF] Rudorfer | Electroconvulsive therapy[END_REF]), (ii) immune inflammation (for details, see [START_REF] Yrondi | Electroconvulsive therapy, depression, the immune system and inflammation: A systematic review[END_REF])) (iii) neuroplastic changes (for details, see (Andrade, 2014a(Andrade, , 2014b;;[START_REF] Taylor | Electroconvulsive therapy, brain-derived neurotrophic factor, and possible neurorestorative benefit of the clinical application of electroconvulsive therapy[END_REF]) and brain functional connectivity changes [START_REF] Mulders | Default mode network coherence in treatment-resistant major depressive disorder during electroconvulsive therapy[END_REF]. Currently, most structural neuroimaging studies focus on changes in hippocampal and amygdala volume after ECT [START_REF] Gbyl | Electroconvulsive therapy increases brain volume in major depression: a systematic review and meta-analysis[END_REF][START_REF] Oltedal | Volume of the Human Hippocampus and Clinical Response Following Electroconvulsive Therapy[END_REF]. Furthermore, most of these studies focus on changes in the volume of these structures between baseline and the end of treatment. Only Joshi et al. [START_REF] Joshi | Structural Plasticity of the Hippocampus and Amygdala Induced by Electroconvulsive Therapy in Major Depression[END_REF] included an MRI after 2 sessions of ECT. In addition, only one study focused on Treatment Resistant Depression (TRD) [START_REF] Gryglewski | Structural changes in amygdala nuclei, hippocampal subfields and cortical thickness following electroconvulsive therapy in treatment-resistant depression: longitudinal analysis[END_REF]. There are a total of only 4 studies that focus on cortical thickness [START_REF] Gryglewski | Structural changes in amygdala nuclei, hippocampal subfields and cortical thickness following electroconvulsive therapy in treatment-resistant depression: longitudinal analysis[END_REF][START_REF] Pirnia | Electroconvulsive therapy and structural neuroplasticity in neocortical, limbic and paralimbic cortex[END_REF][START_REF] Sartorius | Electroconvulsive therapy increases temporal gray matter volume and cortical thickness[END_REF][START_REF] Van Eijndhoven | Bilateral ECT induces bilateral increases in regional cortical thickness[END_REF]. Only one study focused on Treatment Resistant Depression (TRD), but without including comparisons to a control group [START_REF] Gryglewski | Structural changes in amygdala nuclei, hippocampal subfields and cortical thickness following electroconvulsive therapy in treatment-resistant depression: longitudinal analysis[END_REF]. One single study assessed cortical thickness during ECT (after 2 sessions of ECT), even though ECT (with both right unilateral-RUL and bilateral-BL ECT) and diagnosis (21 unipolar disorders/5 bipolar disorders) are heterogeneous [START_REF] Pirnia | Electroconvulsive therapy and structural neuroplasticity in neocortical, limbic and paralimbic cortex[END_REF]. Two other studies only assessed cortical thickness after the treatment [START_REF] Sartorius | Electroconvulsive therapy increases temporal gray matter volume and cortical thickness[END_REF][START_REF] Van Eijndhoven | Bilateral ECT induces bilateral increases in regional cortical thickness[END_REF].

In light of these disparate results in the literature, we aim to assess grey matter changes at different time points during and after ECT BL treatment in patients with TRD (unipolar disorder).

2.Methods

Participants

Patients scheduled to begin ECT at the University Hospital of Toulouse were recruited into the study if they had been diagnosed with TRD (Thase and Rush > =2 [START_REF] Thase | When at first you don't succeed: sequential strategies for antidepressant nonresponders[END_REF]) and were between 50 and 70 years of age. Patient diagnoses were established by clinical consultation using the Diagnostic and Statistical Manual of Mental Disorders 5 (DSM 5) (APA, 2013) criteria with Structured Clinical Interview (SCID-5) [START_REF] First | Structured Clinical Interview for DSM-5 (SCID-5)[END_REF].

Patients with independently diagnosed comorbid psychiatric disorders including schizophrenia, schizoaffective disorders, bipolar disorder, post-traumatic stress disorder, attention hyper-active deficit disorder, dissociative disorders, and patients independently diagnosed with anxiety disorders were excluded from the study. Other exclusion criteria included comorbidity of dementia, Traumatic Brain Injury (TBI), depression related to a medical condition, ECT or other neuromodulation therapies in the previous 6 months, or ECT and anaesthesia contraindications.

Demographically similar healthy controls were recruited from the Toulouse area and screened with the M.I.N.I. to exclude a history of depression, other psychiatric illness, and antidepressant use. Additional exclusion criteria for our patients included a history of alcohol or substance abuse within 6 months and/or dependence within 12 months of participation, neurological disorders, or conditions contraindicated for magnetic resonance imaging (MRI).

The antidepressant treatment was not modified once a patient was included in the study.

Of a total of 17 patients were enrolled in the study. We were able to collect data for all three time points for 15 of these patients (6 females and 9 males) (Table 1). Of the 2 patients who were lost to the study, one continued ECT without MRI assessment and the other discontinued ECT early.

All participants provided written informed consent as required by the CPP Sud Ouest Outre Mer 4 (CPP15-053) (i.e. the 4th ethics research committee of south western France and French overseas territories)

Data acquisition

Patients were assessed clinically and by structural scans (MRI) at three time points: V1 (baseline) completed within 48 hrs prior to the first ECT; V2 completed within the first ECT that was considered effective; and V3 completed within 1 week of completing ECT.

Clinical assessment

The 17-item Hamilton depression rating scale (HAM-D) was administered at each visit to assess symptoms and treatment responses.

ECT

ECT (5000Q MECTA, Tualatin, OR, USA) was administered twice a week, using a standard protocol for anaesthesia (propofol) and paralysis (succinylcholine). ECT followed the seizure threshold (ST) titration method, where following establishment of the ST, treatments were delivered at 1.5 to 2× ST for BL (using brief pulse width). The length of ECT is determined on an individual clinical basis. Patients received a mean of 12.07 ECTs (Table 1).

MRI acquisition and analysis

High-resolution T1-weighted structural scans were acquired using a Philips 3 Tesla (Intera Achieva, Philips, Best, The Netherlands) (170 sagittal slices; scan mode: 3D; multishot; contrast T1; voxel resolution (mm 3 ): 1.00 x 1.00 x 1.00) Structural images were processed using the FreeSurfer's image analysis pipeline (version 5.3.0). To extract reliable volume and thickness estimates, images were automatically processed with the longitudinal pipeline in FreeSurfer [START_REF] Reuter | Within-subject template estimation for unbiased longitudinal image analysis[END_REF]. Specifically, a template space and intra-topic image [START_REF] Reuter | Avoiding asymmetry-induced bias in longitudinal image processing[END_REF] are created using robust coregistration [START_REF] Reuter | Highly accurate inverse consistent registration: A robust approach[END_REF]. Several processing steps, such as skull extraction, Talairach transformations, atlas recording, as well as surface maps and parcellations were then initialised with common information from the internal model, thereby significantly increasing reliability and statistical power [START_REF] Reuter | Within-subject template estimation for unbiased longitudinal image analysis[END_REF]. The automated volumetric segmentation of the grey matter and various structures, including the hippocampus and amygdala, was performed in this manner [START_REF] Fischl | Sequence-independent segmentation of magnetic resonance images[END_REF][START_REF] Fischl | Whole brain segmentation: automated labeling of neuroanatomical structures in the human brain[END_REF]. Segmentations of the various structures were visually reviewed by two independent evaluators (AY and PP). The thicknesses in each region of the FreeSurfer atlas were obtained from FreeSurfer's * .aparc.stats files. The FreeSurfer cortical thickness algorithm calculates the average distance between the vertices of a corrected and triangulated estimated GM/WM surface and the GM/CSF (pial) surface [START_REF] Fischl | Measuring the thickness of the human cerebral cortex from magnetic resonance images[END_REF].

Statistical analysis

Analyses of demographic data and associations between clinical and anatomical data were performed using SPSS (IBM SPSS Statistics for Mac OS, Version 20.0, Armonk, NY, USA).

The Chi2 test was used to compare categorical variables (gender) and the t-test was used to compare continuous variables (age) between groups. A linear regression analysis was used to assess associations between independent variables (delta HAM-D between V1 and V3) and dependent variables (hippocampus and amygdala volume changes). Repeated measures ANOVA was used to compare HAM-D throughout the time series.

Analyses of cortical thickness and subcortical nuclei volumes were performed using R (R Core Team, 2017) and the "nlme" and "lsmeans" packages (Pinheiro et al., 2017).

Cross-sectional data at visit 1 and visit 3 were analysed using mixed-effect linear models. The fixed effect part included cortical thickness or subcortical nuclei volume (normalised for total intracranial volume) as dependent variables and as a group, and regions and their interaction as independent variables. The model also included age and sex in order to correct for any possible effects of these variables on cortical thickness and subcortical nuclei volumes. The random effect included a random intercept for each subject.

CT/Normalised Volume -Baseline/V3ij= β 0 + β 1Group + β 2Frontal Regions/Temporal Regions/Subcortical Nuclei + β3Group X Frontal Regions/Temporal Regions/Subcortical Nuclei + β4Age + β5Sex + u0i + εij
For all significant effects identified, we performed post-hoc comparisons using false discovery rate (FDR) corrections. If the interaction between region and group was significant, we followed up with planned comparisons: by comparing the two groups within each region/nucleus with FDR correction.

Longitudinal data was analysed using mixed-effect linear models. The fixed-effect part included cortical thickness of subcortical nuclei volume (normalised for total intracranial volume) as a dependent variable and visit, group, and their interaction as independent variables. The model also included age and sex in order to correct for any possible confounding effects introduced by these variables.

CT/Normalised Volumeij=β0 + β1Group + β2Visit + β3Group X Visit + β4Age + β5Sex + u0i + εij
In order to reduce the number of comparisons, we analysed the longitudinal data (1) averaged over the sub regions in the frontal and temporal lobe and (2) focused on those regions or subcortical nuclei that differed between patients and controls at baseline. In cases where significant effects were observed, we performed post-hoc comparisons using false discovery rate (FDR) corrections. If the interaction between region and group was significant, we followed up with planned comparisons: by comparing the two groups within each region/nucleus with FDR corrections.

The sub regions included in the frontal and temporal lobes are listed in the supplementary materials.

The threshold for statistical significance was set to 0.05 FDR corrected.

Results

Demographic and clinical data

We analysed 15 patients and 24 healthy controls. Only 18 healthy controls were assessed at all three time points. 6 of these healthy controls only had baseline assessments (MRI). There were no differences between the two populations in terms of age (patients: 59.2 years old (Standard Deviation (SD): 7.1); healthy controls: 61.9 years old (SD :6.9); p=0.921), gender (patients: 6 females/ 9 males; healthy controls: 10 females/ 14 males; p=0.918), and the level of education (patients: 11.57 years (SD :3.27); healthy controls: 12.58 years (SD: 3.94) ; p=0.423).

Clinical data

Using a threshold of a 50% improvement in symptoms at the end of the treatment index, 100% (n=15) of patients were categorised as responsive to treatment. And using a threshold of HAM-D< =7, 93.3% (14 patients) went into remission.

There was an improvement in depressive symptoms after treatment (repeated measures ANOVA for HAM-D(V1)=22.8 (SD=3.05); HAM-D(V2)=20.8 (SD=2.91); HAM-D(V3)=4 (SD=2.65); p< 0.001).

MRI data

Baseline analyses

Frontal lobe sub regions

We detected a significant effect of the group (F[1,35]=13.17, p=< 0.001), while the effect of the region (F[11,407]=0.58, p=0.84) and the interaction between group and region (F[11,407]=1.52, p=0.12) were not significant. Both age and sex were not significant 25, p=0.62). A post-hoc analysis revealed that, at baseline, controls had a greater cortical thickness than patients (t(37)=4.02, p<0.001) (Table 1).

(F[1,35]=2.4, p=0.14; F[1,35]=0.

Temporal Lobe sub regions

The effect of the group was not significant (F[1,35]=1.56, p=0.22), neither was the effect of the region (F[7,259]=1.44, p=0.19). However, the interaction between group and region was found to be significant (F[7,259]=3.74, p<0.001). Age was also found to be significant (F[1,35]=10.48, p=0.002), while sex was not significant (F[1,35]=0.09, p=0.76). A post-hoc analysis revealed that, at baseline, controls had a greater cortical thickness than patients in the fusiform gyrus, the inferior, middle and superior, the parahippocampal gyrus and the transverse temporal gyrus (respectively: t(35)=2.7, p=0.02; t(35)=2.89, p=0.017; t(35)=3.1, p=0.015; t(35)=3.6, p=0.009; t(35)=2.37, p=0.031; t(35)=2.46, p=0.03, FDR corrected), while there were no significant differences in the entorhinal cortex and the temporal pole (all p values > 0.48 FDR corrected). (Table 1.)

Subcortical nuclei

The region played a significant role in the left hemisphere (F[3,111]=454.56, p<0.001), while the effect of the group was not significant (F[1,34]=0.40, p=0.53). The interaction between group and region however, was found to be significant (F[3,111]=4.68, p=0.004). The effect of total grey matter volume was also found to be significant (F[1,34]=42.96, p<0.001), while the effects of age and sex were not significant ((F[1,34]=1.9, p=0.18; (F[1,34]=1.2, p=0.28)).

None of the planned interaction comparisons (i.e., between groups, within regions) were found to be significant (all p values > 0.32).

The region played a significant role in the right hemisphere (F[3,111]=398.62, p<0.001), while the effect of the group was not significant (F[1,34]=0.0, p=0.94). The interaction between group and region was also not significant (F[3,111]=2.55, p=0.059). The effect of total grey matter volume was significant (F[1,34]=69.9, p<0.001), while the effects of age and sex were not found to be significant (F[1,34]=3.35, p=0.076, F[1,34]=3.75, p=0.06)

Visit 3 Analyses

Frontal Lobe sub regions

The effect of the group was not significant and the region was also not found to be significant (respectively F[1,29]=1.7, p=0.2 and F[11,341]=0.27, p=0.99), their interaction was also not significant (F[11,341]=0.59, p=0.83).

Temporal Lobe sub regions

The effect of the group was not significant (F[1,29]=2.32, p=0.14), while the effect of region was found to be significant (F[7,217]=2.54, p=0.016), their interactions were also found to be significant (F[7,217]=5.59, p<0.001). None of the planned interaction comparisons (i.e., between groups, within regions) were found to be significant (all p values > 0.14). (Table 1.)

Subcortical Nuclei

The region played a significant role in the left hemisphere (F[3,93]=357.7, p<0.001), while the effect of the group was not significant (F[1,28]=0.0, p=0.95). The interaction between group and region was not significant (F[3,93]=1.33, p=0.27). The effect of total grey matter volume was significant (F[1,28]=36.5, p<0.001), while age and sex were not significant (F[1,28]=0. 96, p=0.23; F[1,28]=1.8, p=0.19).

The region played a significant role in the right hemisphere (F[3,93]=270.23, p<0.001), while the effect of the group was not significant (F[1,28]=0. 03, p=0.86). The interaction between group and region was not significant (F[3,93]=0.90, p=0.44). The effect of total grey matter volume was also found to be significant (F[1,28]=47.23, p<0.001) while age and sex were not significant (F[1,28]=2.4, p=0.13; F[1,28]=3.74, p=0.06).

Longitudinal Analyses

Frontal Lobe

The effect of the group was significant (F[1,29]=6.49, p=0.016), while the effect of the visit and their interaction were not significant (respectively F[2,62]=0. 78, p=0.46; F [2,62]=1.78 p=0.18). A post-hoc analysis revealed that, at baseline, the controls had a greater cortical thickness than patients (t(29)=2.64 p=0.12).

Temporal Lobe

The effect of the group was significant (F[1,29]=4.7, p=0.038) while the visit was not significant (F[2,62]=0.91, p=0.40). The interaction between group and visit was found to be significant (F[2,62]=4.99, p=0.01). A post-hoc analysis revealed that patients' cortical thickness in the temporal lobe increased between baseline and V3 (t(62)=-2.78 p=0.054 FDR corrected), between V3 and V2 (t(62)=-3.19 p=0. 034 FDR corrected) but did not increase between baseline and V2 (t(62)=-1.63 p=0.4 FDR corrected). Conversely, the different visits had no effect on the controls (all p values > 0.4). Patients were marginally different from controls at baseline (t(29) = 2.2, p=0.065) and significantly different at V2 (t(29) = 2.86, p=0.04), but were not significantly different at V3 (t(29) = p=0.36)

Regions/nuclei differing at V1 -Fusiform Gyrus

The effect of the group was significant (F[1,29]=7.26, p=0.01), while the effect of the visit was not significant (F[2,62]=0.35, p=0.7). The interaction between group and visit was not found to be significant (F[2,62]=1.38, p=0.26).

-Superior Temporal

The effect of the group was significant (F[1,29]=5.74, p=0.02), while the effect of the visit was not significant (F[1,62]=2.2, p=0.12). The interaction between group and visit was found to be significant (F[2,62]=6.82, p=0.002). Post-hoc analysis of the group factor revealed that, on average, controls had a greater cortical thickness than patients, at baseline (t(29)=2.18, p=0.037). Pairwise comparison of the interaction revealed that, cortical thickness in the superior temporal gyrus in the patient group did not significantly increase between baseline and V2 (t(62)=-0.02, p=0.99), but it did significantly increase between V1 and V3 (t(62)=-3.43, p=0.009) and between V2 and V3 (t(62)=-3.42, p=0.009). In contrast, cortical thickness did not increase in the controls (all p values > 0.2). The difference in cortical thickness between controls and patients was significant at baseline and V2 (respectively t(29)=2.40, p=0.054; t(29)=3.7, p=0.004), but was not significant at V3 (t(29)=2, p=0.068) (Figure 1A).

-Middle Temporal

The effect of the group was significant (F[1,29]=4.4, p=0.045), but the effect of the visit was not significant (F[1,29]=1.59, p=0.21). The interaction between group and visit was also not found to be significant (F[2,62]=3, p=0.057).

-Parahippocampal gyrus

The effects of the group and the visit were not significant (respectively F[1,29]=0.2, p=0.17; F[2,62]=0.1, p=0.9), and their interaction was also not significant (F[2,62]= 2.75, p=0.071).

-Transverse occipital gyrus

The effect of the group was significant (F[1,29]=5.92, p=0.021), while the visit was not significant (F[2,62]=1. 87, p=0.16). The interaction between group and visit was also not found to be significant (F[2,62]=0.35, p=0.70).

-Hippocampus

The effects of the group and the visit were not significant (respectively F[1,31]=0.19, p=0.66; F[2,61]=1.47, p=0.24). However, the interaction between group and visit was significant (F[2,62]=15.3, p<0.001). A post-hoc analysis revealed that in the patient group, cortical thickness in the hippocampus did not increase between baseline and 2 (t(62)=-0.44, p=0.63), but it did significantly increase between V1 and V3 (t(62)=-5.23, p<0.001) and between V2 and V3 (t(62)=-5.3, p<0.001). ). In contrast, cortical thickness did not increase in the controls (all p values > 0.65). A significant difference between patients and controls was detected at all the time points examined (all p values > 0.65). (Figure 1B) -Amygdala

The effects of the group, the visit, and their interaction were not significant (respectively 96, p=0.39; F[2,61]=2.71, p=0.074). The effect of total grey matter volume was significant (F[1,61]=33.46, p=< 0.001).

F[1,29]=0.2, p=0.64; F[2,61]=0.

-Caudate

The effects of the group, the visit, and their interaction were not significant (respectively F[1,29]=2.3, p=0.14; F[2,61]=0.06, p=0.93; F[2,61]=0.5, p=0.61). The effect of total grey matter volume was significant (F[1,61]=17, p=< 0.001).

-Putamen

The effects of the group, the visit, and their interaction were not significant (respectively F[1,29]=1.37, p=0.25; F[2,61]=0.4, p=0.67; F[2,61]=0.46, p=0.63). The effect of total grey matter volume was significant (F[1,61]=31.3, p=< 0.001).

Effect of clinical response

There was no significant association between structural MRI changes and clinical improvement (Delta HAM-D between V1 and V3): (i) Hippocampus (β=0.245; p=0.38) and (ii) Amygdala (β=-0.256; p=0.357)

Discussion

A baseline comparison of a population of patients with TRD and healthy controls detected a significantly greater cortical thickness in the controls in the fusiform gyrus, in the parahippocampal gyrus, and in the transverse, middle and superior temporal gyrus. These differences were no longer significant after the whole sessions of ECTs. Moreover, we detected a significant increase in cortical thickness in the superior temporal gyrus in the patient group between (i) baseline and V3 and (ii) V2 and V3, but not between baseline and V2. This suggests that grey matter changes only occur after multiple ECT sessions.

In addition to changes in cortical thickness, we observed an increase in hippocampal volume between (i) baseline and V3 and (ii) V2 and V3 in our patient group. These results parallel those found for cortical thickness, suggesting that grey matter volume only increases after multiple sessions of ECT. We did not detect any changes in the amygdala, caudate, or putamen.

As suggested by Pirnia et al. [START_REF] Pirnia | Electroconvulsive therapy and structural neuroplasticity in neocortical, limbic and paralimbic cortex[END_REF] and [START_REF] Gryglewski | Structural changes in amygdala nuclei, hippocampal subfields and cortical thickness following electroconvulsive therapy in treatment-resistant depression: longitudinal analysis[END_REF], we showed an increase in cortical thickness in the superior temporal gyrus in patients with TRD treated using ECT (and not in controls). Our results are in agreement with the current literature [START_REF] Gbyl | Electroconvulsive therapy increases brain volume in major depression: a systematic review and meta-analysis[END_REF][START_REF] Oltedal | Volume of the Human Hippocampus and Clinical Response Following Electroconvulsive Therapy[END_REF][START_REF] Yrondi | Structural-functional brain changes in depressed patients during and after electroconvulsive therapy: a systematic review of magnetic resonance imaging studies[END_REF], as we detected an increase in hippocampal volume. Our results nevertheless suggest that these changes only occur after several effective sessions of ECT. Indeed, we did not find any differences between baseline and the first assessment in hippocampal nor cortical thickness. It seemed that we cannot discount a cumulative effect of ECT on these brain structures, since we were unable to detect effects of a single ECT session, perhaps because this effect may have be too weak to be detected in our sample.

To our knowledge, this is the first study to monitor differences in grey matter (cortical thickness and subcortical region) at 3 distinct time points in a population of patients with TRD treated with ECT BL, compared to a healthy control population. As it is likely that the majority of patients treated with ECT for MDD probably have TRD, our results can therefore be compared to many others studies focusing on MDD, but which did not specifically examine treatment resistant depression. The consortium Global ECT-MRI Research Collaboration (GEMRIC ) find that volume enlargement is not significantly related to treatment outcome. This is consistent with our results specifically in our TRD population. Indeed, we did not find any significant association between structural MRI changes and clinical improvement. However, contrary to our results, Oltedal et al. (GEMRIC) [START_REF] Oltedal | The Global ECT-MRI Research Collaboration (GEMRIC): Establishing a multi-site investigation of the neural mechanisms underlying response to electroconvulsive therapy[END_REF] show an inverse relationship between hippocampal volume and the improvement of symptoms.

In addition to the Gbyl's meta-analysis [START_REF] Gbyl | Electroconvulsive therapy increases brain volume in major depression: a systematic review and meta-analysis[END_REF], recent studies seem to confirm this increase of hippocampal and amygdala volume in MDD populations. In the largest study (N=92) examining the effect of ECT on the brain structure of an MDD population (not specifically resistant), Sartorius et al. [START_REF] Sartorius | Electroconvulsive therapy induced gray matter increase is not necessarily correlated with clinical data in depressed patients[END_REF] show an increase in temporo-mesial regions mainly in the hippocampus and amygdala. When examining subfields of hippocampus, Nuninga et al. [START_REF] Nuninga | Volume increase in the dentate gyrus after electroconvulsive therapy in depressed patients as measured with 7T[END_REF] report a large and significant increase in Dentate Gyrus volume (MRI 7 tesla) after ten bilateral ECT sessions. Moreover, [START_REF] Bouckaert | Relationship Between Hippocampal Volume, Serum BDNF, and Depression Severity Following Electroconvulsive Therapy in Late-Life Depression[END_REF] also detected an increase in hippocampal volume after ECT in specific populations (N=88) with late life depression (>55 years old). The age of this population is comparable to our population and, even though this was not a specific TRD population, these results are consistent with ours.

In light of the results reported in the literature, the strengths of our study are (i) an MRI scan very early (after the first ECT session that was considered effective) which suggests that there is a cumulative effect of ECT on brain structure; (ii) a population limited to patients ranging from 50 to 70 years of age (to select a more homogeneous population in terms of aging brain structures) with only unipolar TRD (to limit the heterogeneity of our sample) and (iii) exclusion criteria including anxiety disorder and PTSD (to limit the heterogeneity of our sample). However, although this is a population with severe TRD according to the thresholds recommended in the APA's Handbook of Psychiatric Measures [START_REF] Rudorfer | Electroconvulsive therapy[END_REF] to define grades of severity on the HAM-D: mild to moderate: 8-18; severe: 19-22; very severe ≥23 (ranging from 19 to 29 in our sample) and according to the thresholds recommended in Thase and Rush scale to define different grades of resistance: ≥2 (Thase and Rush, 1997) (ranging from 2 to 4 in our sample), the severity and the resistance could be considered as relatively low with, in addition to this, 2 attempted suicides and 2 lifetime hospitalisations for depressive disorders. Moreover, the patients are rather old (50-70 years), which is a prognostic response factor [START_REF] Van Diermen | Prediction of electroconvulsive therapy response and remission in major depression: meta-analysis[END_REF]. In addition to this, patients do not have any psychiatric comorbidity. Finally, patients received a large number of ECTs [12.1 (5.26)].

These data could explain the very high response and remission rates in our sample. All these issues should be taken into account when extrapolating from our results.

Limitations

Our study does have some limitations. Firstly, we included only a small number of patients (15), which precluded the analyses of different MDD clusters [START_REF] Drysdale | Resting-state connectivity biomarkers define neurophysiological subtypes of depression[END_REF]. Thus, this increases the risk of a heterogeneous population. Moreover, we decided to maintain the antidepressant treatment during ECT (as we do in daily care in our unit). This can affect grey matter changes if we take the effects of antidepressants into consideration. But to limit this effect, we did not alter antidepressant treatment after inclusion of patients into the study. Moreover, our last visit was close to the end of the course of ECT. However, some studies report a decrease in the volume of brain structures such as the hippocampus throughout the time following the treatment without a relapse in depressive symptoms [START_REF] Bouckaert | Relationship Between Hippocampal Volume, Serum BDNF, and Depression Severity Following Electroconvulsive Therapy in Late-Life Depression[END_REF][START_REF] Nordanskog | Hippocampal volume in relation to clinical and cognitive outcome after electroconvulsive therapy in depression[END_REF]. These data should be taken into account when interpreting our results.

Perspectives

Future studies with larger sample sizes and/or consortium initiatives such as the GEMRIC [START_REF] Oltedal | The Global ECT-MRI Research Collaboration (GEMRIC): Establishing a multi-site investigation of the neural mechanisms underlying response to electroconvulsive therapy[END_REF] seem to be necessary to better understand the mechanisms underlying ECT effects. Moreover, assessment of mean diffusivity and fractional anisotropy (in Diffusion Tensor Imaging) of hippocampus in addition to volume could be of interest to understand these mechanisms and potentially assess a link between volumetric increases and treatment effects and side effects.

Conclusion

We report grey matter changes during ECT in a population with TRD when compared to a healthy control population. These changes seem to occur after multiple ECT sessions. This

Figure 1 .

 1 Figure 1. Longitudinal change in superior temporal gyrus (A) and Hippocampus (B) over time

Table 1 .

 1 Degrees of Freedom (DF). statistics and significance of the cross-sectional and longitudinal statistical analyses. Main effect and interactions are in plain text. post-hoc analyses are underlined. HC = Healthy Controls. PT = Patients. 1 F statistic is reported for main effects and interactions while t statistic is reported for post-hoc comparisons. 2 Uncorrected p values are reported for main effects and interactions. FDR corrected p values are reported for post-hoc comparisons

	t 1	p 2

Acknowledgements: This work was supported by a grant from the Délégation Régionale à la Recherche Clinique des Hôpitaux de Toulouse "2015".

We thank the Clinical Investigation Centre in Toulouse (Calvas F. Galitski M), the technical platform for neuroimaging (TONIC: Gros H. Vayssiere N.) and HoPes: FHU (Fédération Hospitalo-Universitaire) devoted to "Cognitives, Psychic and Sensorial disabilities" (Méligne D.)

may contribute to a better understanding of the ECT mechanism.

Conflicts of interest disclosure

The authors declare no competing financial interests.


Van-Thuan Hoang

Doudou Sow

Fabiola Dogue

Tassadit Drali

Saber Yezli

Badriah Alotaibi

Didier Raoult

Philippe Parola

Vincent Pommier De Santi

Philippe Gautret
email: philippe.gautret@club-internet.fr

Acquisition of respiratory viruses and presence of respiratory symptoms in French pilgrims during the 2016 Hajj: A prospective cohort study

Keywords: Hajj, mass gathering, respiratory tract infection, viruses, PCR

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

Introduction

Hajj pilgrimage has long been associated with enhanced transmission of infectious disease agents. Epidemics of cholera [START_REF] Ahmed | Health risks at the Hajj[END_REF] and bacterial meningitis [START_REF] Yezli | Meningococcal disease during the Hajj and Umrah mass gatherings[END_REF][START_REF] Yezli | The threat of meningococcal disease during the Hajj and Umrah mass gatherings: A comprehensive review[END_REF] are emblematic examples of the potential for international spread of life-threatening infections at the Hajj, given its international component with pilgrims originating from up to 180 countries and gathering in Mecca before returning to their home country [START_REF] Memish | Hajj: infectious disease surveillance and control[END_REF]. More recently, respiratory tract infections at the Hajj have attracted the attention of the medical community because of the frequency of respiratory symptoms among pilgrims consulting at primary health care facilities or hospitalized in Saudi Arabia [START_REF] Al-Tawfiq | Respiratory tract infections during the annual Hajj: potential risks and mitigation strategies[END_REF]. Cohort studies conducted in populations of pilgrims originating from different countries reported that a substantial proportion of pilgrims suffer from respiratory symptoms during their stay in Saudi Arabia [START_REF] Al-Tawfiq | Mass gatherings and the spread of respiratory infections. Lessons from the Hajj[END_REF]. On the other hand, numerous PCR-based studies have demonstrated the frequent acquisition of respiratory viruses following participation in Hajj [START_REF] Gautret | Hajj-associated viral respiratory infections: A systematic review[END_REF][START_REF] Al-Tawfiq | A systematic review of emerging respiratory viruses at the Hajj and possible coinfection with Streptococcus pneumoniae[END_REF]. Fortunately, SARS-CoV and MERS-Cov did not affect Hajj pilgrims so far [START_REF] Al-Tawfiq | Travel implications of emerging coronaviruses: SARS and MERS-CoV[END_REF][START_REF] Gautret | The spectrum of respiratory pathogens among returning Hajj pilgrims: myths and reality[END_REF]. However, rhinovirus, non-MERS coronaviruses and influenza viruses are commonly isolated from both asymptomatic returning pilgrims and pilgrims with acute respiratory symptoms [START_REF] Gautret | Hajj-associated viral respiratory infections: A systematic review[END_REF][START_REF] Al-Tawfiq | A systematic review of emerging respiratory viruses at the Hajj and possible coinfection with Streptococcus pneumoniae[END_REF]. Due to the lack of detailed clinical information in many Hajj studies and the high sensitivity of PCR tools, the contribution of viruses to observed symptoms remains unknown.

The objective of the study was to evaluate the nasal carriage of respiratory viruses before and after traveling to the Hajj, and to investigate a possible relationship between viral carriage and respiratory symptoms with a careful clinical follow-up in a cohort of French Hajj pilgrims departing from Marseille.

Materials and methods

Study population

The Figure 1 details the procedure of this study. The study was conducted among French Hajj pilgrims travelling together to Mecca, from August 27 th to September 20 th , 2016, with one specialized travel agency in Marseille. Pilgrims older than 18 years were included on a voluntary basis, and participants were asked to sign a written consent form. Upon inclusion, the participants were questioned using a standardized pre-travel questionnaire that included demographic and chronic disease data and vaccination status. Health issues were recorded by a medical doctor who travelled with the group of pilgrims. We considered that participants suffered RTIs if they presented with cough and/or rhinitis and/or sore throat. Influenza like illness (ILI) was defined as the association of cough, sore throat and fever [START_REF] Rashid | Influenza and the Hajj: defining influenza-like illness clinically[END_REF]. Each individual was classified in one of the three categories: i. asymptomatic (those who did not experience any respiratory symptoms during the entire stay in Saudi Arabia), ii. resolved respiratory tract infection (RTI) (those who experienced respiratory symptoms including cough and/or sore throat and/or rhinitis and/or voice failure during travel, but who recovered at the time of return to France and iii. ongoing RTI (those with ongoing respiratory symptoms at the time of leaving Saudi Arabia).

Sample collection

The procedure included a systematic nasal swab 10 days before departing from France (pre-Hajj specimens) and just 1 day (on September 19 th , 2016) before leaving the KSA (Kingdom of Saudi Arabia) (post-Hajj specimens) We previously showed that nasal swabs are more sensitive than pharyngeal swabs in detecting respiratory viruses in Hajj pilgrims, using realtime reverse transcriptase-polymerase chain reaction methods [START_REF] Benkouiten | Comparison of nasal swabs with throat swabs for the detection of respiratory viruses by real-time reverse transcriptase PCR in adult Hajj pilgrims[END_REF]. Nasal swabs were also performed among symptomatic pilgrims who spontaneously consulted the accompanying medical doctor at the time of onset (per-Hajj specimens). No per-Hajj sample was collected among asymptomatic pilgrims. Samples were collected using commercial rigid cotton-tipped swab applicators (Medical Wire & Equipment, Wiltshire, UK) which were inserted in the anterior nose and then placed in viral transport media (Sigma Virocult ® ). This standardized procedure was previously explained to the pilgrims by the investigators. The swabs were stored at 20°C before being transported to the Marseille laboratory for storage in a freezer at -80°C within 48 hours of collection for pre-and post-Hajj samples. Per-Hajj specimen were kept at 20°C until the return to France.

Identification of respiratory pathogens

The analyses were carried out in Marseille, following return (thus results were not available during the stay in Saudi Arabia) with a validated multiplex qualitative PCR method [START_REF] Leber | Multicenter Evaluation of BioFire FilmArray Respiratory Panel 2 for Detection of Viruses and Bacteria in Nasopharyngeal Swab Samples[END_REF]. The BioFire respiratory panel (BFRP, BioFire) includes the following virus targets: rhinovirus/enterovirus, adenovirus, human coronavirus (229E, HKU1, NL63 and OC43), human metapneumovirus, influenza virus A and B, parainfluenza virus (1, 2, 3 and 4) and respiratory syncytial virus. Three bacteria are targeted in the test including Bordetella pertussis, Chlamydia pneumoniae and Mycoplasma pneumoniae. Acquisition of pathogens was defined as the absence of a given pathogen in pre-Hajj samples and the presence of this pathogen in per-and or post-Hajj samples.

Statistical analysis

The Pearson's Chi-square test and Fisher's exact test, as appropriate, were applied to analyze the categorical variables. To evaluate the potential acquisition of respiratory viruses in Saudi Arabia, we used the McNemar's Test to compare their percentage before leaving France and in Saudi Arabia. Percentages and odds ratio (OR) with 95% confidence interval (CI) estimations and comparisons were carried out using the STATA 11.1 (Copyright 2009 StataCorp LP, http://www.stata.com). P values of .05 or less were considered significant.

Results

Demographics, and respiratory symptoms

A total of 109 pilgrims were included on departure from France. The median age was 63 years (interquartile: 55-69, range: 23-96), and 46.8% were males. 11 (10.1%) individuals reported a history of chronic respiratory disease and only one pilgrim had respiratory symptoms before leaving Marseille. At total, 26/109 (23.8%) pilgrims received influenza vaccination during the past 12 months, but only 2/109 (1.8%) reported having been vaccinated against pneumococcal (PCV-13) in the last 5 years. A total of 91 (83.5%) pilgrims presented at least one respiratory symptom during their stay in Saudi Arabia with cough, rhinitis and sore throat being most frequent (Table 1). The median time between arrival to Saudi Arabia and symptom onset was 9 days (range: 0-18 days). The majority of pilgrims had their onset of symptoms during their stay in Mecca and Mina (Figure 2). Twenty-five (22.9%) pilgrims had influenza like illness. 30 (27.5%) pilgrims took antibiotics for the purpose of respiratory tract infection symptoms, based on the clinical judgment of the accompanying doctor and none was hospitalized. Fortythree (39.5%) were still symptomatic at the time of leaving Saudi Arabia.

Acquisition of virus and bacteria carriage

Nasal swabs were obtained from 109 pilgrims (100%) before traveling to Saudi Arabia, from 21 patients at symptom onset in Saudi Arabia and from 99 (90.8%) pilgrims before leaving Saudi Arabia for France. Among these, 99 individuals (90.8%) underwent paired samples allowing the calculation of acquisition rates. The proportion of pilgrims positive for each potential pathogen detected is presented in Table 2. Of the pre-Hajj samples, 6 (5.5%) were positive, while a pathogen was found in 20 (95.2%) per-Hajj samples and in 46 (46.5%) post-Hajj samples (p<0.0001). Overall, the acquisition rate of at least one pathogen during the stay in Saudi Arabia was 40.0%. The proportion of pilgrims positive for rhinovirus/enterovirus and coronavirus 229E were significantly higher in post-Hajj samples, compared to pre-Hajj samples, with acquisition rates of respectively 38.6% and 19.8%. Multiple infections were frequent accounting for 50.0% positive per-Hajj samples and 17.4% of positive post-Hajj samples with rhinovirus/enterovirus-coronavirus 229E mixed infection the most common (Table 3). No case was positive for the three bacteria tested.

Virus carriage according to respiratory symptoms

Among 108 asymptomatic pilgrims sampled in France before travel, 5/108 (4.6%) were positive for rhinovirus/enterovirus. The only pilgrim suffering respiratory symptoms before travel was positive for rhinovirus/enterovirus. Among the 21 pilgrims sampled at symptom onset in Saudi Arabia, 20 tested positive for at least one virus with high proportion of rhinovirus/enterovirus (81.0%), coronavirus 229E (33.0%) and influenza A (23.8%) (Table 2). Seven of the 21 symptomatic pilgrims had ILI and their carriage rate did not significantly differ from that of other ill pilgrims (data not shown). Of the 21 pilgrims sampled at the time of symptom onset, 19 were resampled before leaving Saudi Arabia, allowing the proportion of individuals who allowed their viral transport back to the country to be calculated. The mean time between onset of symptoms and testing on leaving KSA was 15.4 days (ranging 8-19). Rhinovirus/enterovirus carriage was cleared in 70.6.1% cases, coronavirus 229E carriage in 71.4% cases and influenza virus A carriage in 100% cases, while the majority of cases were still symptomatic (71.4%) (Figure 3).

Comparison of virus carriage at the time of return, according to the presence of respiratory symptoms at sampling time showed a highest proportion of virus positivity (at least one virus) in symptomatic patients (57.9%) compared to those who have never experienced cough or who resolved it (39.3%), but the difference was not significant (OR = 2.11, 95% CI[0.93 -4.83] (Tables 4). However, carriage of at least one virus on return was significantly higher in patients with ongoing ILI symptoms (75.0%) compared to patients without ILI symptoms or with resolved ILI symptoms (42.5%), OR = 4.05, 95%CI [1.02 -16.02] (Table 5). Breakdown by virus type; however, did not show statistically significant variations, although their percentage of positivity was higher in pilgrims presenting with at least one respiratory symptom or ILI symptoms at the time of sampling, in comparison with those who did not.

Comparison of virus carriage in patients who experienced symptoms during the Hajj (ongoing and resolved) and in patients who were asymptomatic at any time did not show significant differences.

Discussion

We observed both a high frequency of respiratory symptoms during the pilgrims' stay in KSA (76.2% cough and 22.9% ILI) and a significant acquisition of viral nasal carriage (40.0%), mostly due to rhinovirus/enterovirus and coronavirus 229E. This corroborates the results obtained from most recent studies conducted among French pilgrims by our team and among pilgrims from other nationalities [START_REF] Benkouiten | Circulation of respiratory viruses among pilgrims during the 2012 Hajj pilgrimage[END_REF][START_REF] Benkouiten | Respiratory viruses and bacteria among pilgrims during the 2013 Hajj[END_REF][START_REF] Rashid | Viral respiratory infections at the Hajj: comparison between UK and Saudi pilgrims[END_REF][START_REF] Memish | Detection of respiratory viruses among pilgrims in Saudi Arabia during the time of a declared influenza A(H1N1) pandemic[END_REF][START_REF] Barasheed | Viral respiratory infections among Hajj pilgrims in 2013[END_REF][START_REF] Memish | Mass gathering and globalization of respiratory pathogens during the 2013 Hajj[END_REF][START_REF] Annan | High prevalence of common respiratory viruses and no evidence of Middle East respiratory syndrome coronavirus in Hajj pilgrims returning to Ghana, 2013[END_REF][START_REF] Atabani | Active screening and surveillance in the United Kingdom for Middle East respiratory syndrome coronavirus in returning travellers and pilgrims from the Middle East: a prospective descriptive study for the period 2013-2015[END_REF][START_REF] Alnabulsi | Social identification moderates the effect of crowd density on safety at the Hajj[END_REF]. The epidemic curves of onset of symptoms and virus carriage in samples obtained at onset of symptoms suggest an early acquisition of respiratory virus during the initial stay in Mecca, probably due to inter-human transmission as attested by the bi-modal pattern of the curves during this period of time. Crowded conditions at Al-Haram Mosque during the rituals, with up to 8 individuals per m 2 recorded close to the Kaaba [START_REF] Kandeel | Pandemic (H1N1) 2009 and Hajj Pilgrims who received Predeparture Vaccination, Egypt[END_REF], is likely to play a significant role in this process. Asymptomatic carriage of rhinovirus/enterovirus and coronaviruses was frequently observed when tested in returning pilgrims. Nevertheless, overall viral carriage in patients with ILI on return (75.0%) was significantly higher than in individuals without ILI (42.5%), which suggests that viruses play a role in the pathogenesis of the RTIs. Viruses were detected in almost all 21 pilgrims sampled at symptom onset, which reinforces this view. However, while most of these 21 pilgrims were still symptomatic at the post-Hajj sampling time; only a low proportion of those testing positive at onset remained positive in post-Hajj samples two weeks after first symptoms. These results, although based on small numbers of ill pilgrims clearly demonstrate that sampling at the time of leaving KSA results in underestimation of viral carriage in relation with symptoms during the stay because the majority of ill pilgrims already cleared their viral infection despite persisting symptoms, as observed in our survey. Since obtaining respiratory samples at onset of symptoms is challenging in the context of longitudinal cohort survey at the Hajj, most studies conducted so far were based on systematic samples obtained at the time of return, days after the onset of symptoms occurred. Such a design evaluating viral carriage at return provides useful on the potential for respiratory viruses to spread upon return to the country of origin, assuming that some of the viruses detected by PCR are viable.

It is however less appropriate for retrospectively investigating the responsibility of viruses in respiratory symptoms experienced during the stay in Saudi Arabia, given the rapid virus clearance. This was particularly obvious in our survey with a proportion of influenza A virus positivity of 2.0% in pilgrims screened on return, regardless of symptoms contrasting with 20% in pilgrims who actively consulted our medical investigator sampled at respiratory symptom onset. Studies conducted in different countries from 2009 through 2015 with the aim of screening influenza virus by PCR among returning pilgrims regardless of symptoms, reported a mean influenza carriage rate of 3.4%, ranging 0.4-7.8% [START_REF] Benkouiten | Circulation of respiratory viruses among pilgrims during the 2012 Hajj pilgrimage[END_REF][START_REF] Benkouiten | Respiratory viruses and bacteria among pilgrims during the 2013 Hajj[END_REF][START_REF] Memish | Mass gathering and globalization of respiratory pathogens during the 2013 Hajj[END_REF][START_REF] Kandeel | Pandemic (H1N1) 2009 and Hajj Pilgrims who received Predeparture Vaccination, Egypt[END_REF][START_REF] Memish | Detection of respiratory viruses among pilgrims in Saudi Arabia during the time of a declared influenza A(H1N1) pandemic[END_REF][START_REF] Ziyaeyan | Pandemic 2009 influenza A (H1N1) infection among 2009 Hajj Pilgrims from Southern Iran: a real-time RT-PCR-based study[END_REF][START_REF] Ma | No MERS-CoV but positive influenza viruses in returning Hajj pilgrims, China, 2013-2015[END_REF], which is in line with the results reported here (1.9%). When similar screening studies were conducted, enrolling only pilgrims with respiratory symptoms on returning to their home country, a higher proportion of 10.6% influenza carriage was observed, ranging 3.0-14.5% when we found a slightly lower proportion of 2.1% in this study [START_REF] Moattari | Influenza viral infections among the Iranian Hajj pilgrims returning to Shiraz, Fars province, Iran[END_REF][START_REF] Refaey | Cross-sectional survey and surveillance for influenza viruses and MERS-CoV among Egyptian pilgrims returning from Hajj during 2012-2015[END_REF][START_REF] Koul | Influenza not MERS CoV among returning Hajj and Umrah pilgrims with respiratory illness, Kashmir, north India, 2014-15[END_REF][START_REF] Rha | Acute respiratory infections among returning Hajj pilgrims-Jordan, 2014[END_REF][START_REF] Muraduzzaman | Event based surveillance of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Bangladesh among pilgrims and travelers from the Middle East: An update for the period 2013-2016[END_REF]. Finally, in studies conducted in sick pilgrims seen in Saudi or European hospitals for suspected MERS-Cov infection and therefore suffering from more severe respiratory symptoms, the percentage of confirmed influenza infection was 34.9%, ranging from 12.0 to 71.4% [START_REF] Atabani | Active screening and surveillance in the United Kingdom for Middle East respiratory syndrome coronavirus in returning travellers and pilgrims from the Middle East: a prospective descriptive study for the period 2013-2015[END_REF][START_REF] Aberle | Influenza A and B Viruses but Not MERS-CoV in Hajj Pilgrims, Austria, 2014[END_REF][START_REF] Griffiths | Infections in symptomatic travelers returning from the Arabian peninsula to France: A retrospective cross-sectional study[END_REF][START_REF] Yavarian | Influenza virus but not MERS coronavirus circulation in Iran[END_REF][35].

Our study has some limitations. First, it was conducted among a small number of pilgrims during one season of Hajj, and our results cannot be extrapolated to all pilgrims. Secondly, we were able to collect respiratory samples at symptom onset in only one fourth of patients who reported suffering respiratory symptoms during the study period. This low proportion is due to the nature of the recruitment since sampling at onset of symptoms was not systematic but performed in patients who spontaneously consulted the accompanying doctor at the early beginning of their illness. The sample type used is not FDA cleared or CE marked and the BFRP, BioFire performance characteristics using nasal swabs instead of nasopharyngeal swabs have not been established.

Conclusion

Viruses are acquired by the vast majority of Hajj pilgrims soon after their arrival in Mecca and likely responsible for respiratory symptoms, notably ILI. Viral clearance is rapid. Point of care-rapid multiplex PCR assays are valuable diagnosis tools for Hajj patients when used at respiratory symptom onset or soon after. In particular, they allow the detection of the influenza virus, which is particularly interesting because it has practical consequences for the early prescription of antivirals in people at risk. These tests are also useful for ruling-out MERC-CoV infection and deciding about isolation measure lifting. *at least one respiratory symptom (cough, sore throat, rhinitis)

OR: odds ratio, CI: confidence interval

  The protocol was approved by our Institutional Review Board (July 23, 2013; reference No.2013-A00961-44). It was performed in accordance with the good clinical practicesrecommended by the Declaration of Helsinki and its amendments. All participants gave a written informed consent.

Figure 1 :

 1 Figure 1: Flow diagram of study participants

Figure 2 .

 2 Figure 2. Respiratory symptom onset date in 91 ill pilgrims

Figure 3 :

 3 Figure 3: Respiratory virus carriage among pilgrims sampled at symptom onset and on return

Table 2 .

 2 Frequency of pathogens detected in pre-, per-and post-Hajj samples

Table 3 .

 3 Frequency of carriage of multiple pathogen combinations

Table 4 .

 4 Frequency of pathogens in post-Hajj samples according to respiratory symptoms at the time of sampling.

Table 5 .

 5 Frequency of pathogens in post-Hajj samples according to influenza-like illness symptoms at the time of sampling.

Table 1 .

 1 Clinical symptoms in 109 participants

	Symptoms

Table 4 .

 4 Frequency of pathogens in post-Hajj samples according to respiratory symptoms at the time of sampling.

			Asymptomatic	Resolved	Ongoing	OR [95%CI], P value	OR [95%CI], P value
			(N = 16)	symptoms*	symptoms*	(ongoing symptoms versus	(ongoing or resolved symptoms
				(N = 45)	(N = 38)	asymptomatic or resolved symptoms)	versus asymptomatic)
	Rhinovirus/enterovirus Yes	5 (31.3)	13 (28.9)	14 (36.8)
						1.39 [0.59 -3.29], P = 0.45	1.06 [0.34 -3.36], P = 0.92
		No	11 (68.7)	32 (71.1)	24 (63.2)
	Adenovirus	Yes	0 (0)	1 (2.2)	2 (5.3)
						3.33 [0.30 -38.08], P = 0.33	-
		No	16 (100)	44 (97.8)	36 (94.7)
	Coronavirus 229E	Yes	0 (0)	8 (17.8)	7 (18.4)
						1.50 [0.49 -4.53], P = 0.48	-
		No	16 (100)	37 (82.2)	31 (81.6)
	Coronavirus HKU1	Yes	0 (0)	0 (0)	1 (2.6)
						-	-
		No	16 (100)	45 (100)	37 (97.4)
	Coronavirus NL63	Yes	0 (0)	0 (0)	1 (2.6)
						-	-
		No	16 (100)	45 (100)	37 (97.4)
	Coronavirus OC43	Yes	0 (0)	0 (0)	2 (5.3)
						-	-
		No	16 (100)	45 (100)	36 (94.7)
	Influenza A	Yes	0 (0)	1 (2.2)	1 (2.6)	1.62 [0.10 -26.72], P = 0.74	-

Acknowledgement

This work was supported by bioMérieux and by the Agence Nationale de la Recherche (programme "investissement d'avenir" Méditerranée Infection 10-IAHU-03), the Région Provence-Alpes-Côte d'Azur and European funding FEDER PRIMI.


Van-Thuan Hoang

Thi-Thu-Thuy Nguyen

Khadidja Belhouchat

Mohammed Meftah

Doudou Sow

Samir Benkouiten

Thi-Loi Dao

Tran Duc

Anh Ly

Tassadit Drali

Saber Yezli

Badriah Alotaibi

Didier Raoult

Philippe Parola

Vincent Pommier De Santi

Philippe Gautret
email: philippe.gautret@club-internet.fr

Antibiotic use for respiratory infections among Hajj pilgrims: A cohort survey and review of the literature

Keywords: Hajj, pilgrims, antibiotic, respiratory tract infections, recommendation, determinants

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

Introduction

The Muslim Hajj pilgrimage in Saudi Arabia, is among the largest annual religious mass gatherings on earth. Each year, it welcomes more than 2 million pilgrims from more than 180 countries, a third of whom come from outside Saudi Arabia. The Hajj takes place in three main places in the Mecca area, which are the Grand Mosque in the Holy city of Mecca, the Mina valley and the plain of Arafat (about 5 and 18 kilometers from Mecca respectively) [START_REF] Gatrad | Hajj: journey of a lifetime[END_REF]. A large proportion of pilgrims are elderly people, many with chronic diseases [START_REF] Memish | Hajj: infectious disease surveillance and control[END_REF]. Furthermore, the presence of many pilgrims from different countries around the world and overcrowding considerably increase the risk to contract infectious diseases, particularly respiratory and gastrointestinal infections, resulting in a considerable demand for antibiotic use [START_REF] Abubakar | Global perspectives for prevention of infectious diseases associated with mass gatherings[END_REF][START_REF] Al-Tawfiq | Mass gatherings and infectious diseases: prevention, detection, and control[END_REF]. The predominance of bacterial pathogens in Hajj-related gastrointestinal infections poses a major public health risk due to the potential emergence and transmission of antimicrobial resistant bacteria [START_REF] El Ghany | Enteric Infections Circulating during Hajj Seasons, 2011-2013[END_REF]. Antibiotic resistant gastro-intestinal and respiratory organisms have been frequently isolated from Hajj pilgrims [START_REF] Asghar | Frequency and antimicrobial susceptibility patterns of bacterial pathogens isolated from septicemic patients in Makkah hospitals[END_REF][START_REF] Asghar | Frequency and antimicrobial susceptibility of gram negative bacteria isolated from 2 hospitals in Makkah, Saudi Arabia[END_REF][START_REF] Leangapichart | Risk factors for acquisition of CTX-M genes in pilgrims during Hajj 2013 and 2014[END_REF][START_REF] Leangapichart | Acquisition of extended-spectrum b-lactamases by Escherichia coli and Klebsiella pneumoniae in gut microbiota of pilgrims during the hajj pilgrimage of 2013[END_REF][START_REF] Leangapichart | Acquisition of a high diversity of bacteria during the Hajj pilgrimage, including Acinetobacter baumannii with blaOXA-72 and Escherichia coli with blaNDM-5 carbapenemase genes[END_REF][START_REF] Leangapichart | Acquisition of mcr-1 plasmid-mediated colistin resistance in Escherichia coli and Klebsiella pneumoniae during Hajj 2013 and 2014[END_REF]. Although a prescription has been required in order to receive antibiotics in Saudi Arabia for more than 30 years [START_REF] Bawazir | Prescribing pattern at community pharmacies in Saudi Arabia[END_REF], many pilgrims use antibiotic without a prescription [START_REF] Azeem | Knowledge, Attitude and Practice (KAP) Survey Concerning Antimicrobial Use among Australian Hajj Pilgrims[END_REF]. To date, few studies on the appropriateness of antibiotic use among pilgrims were conducted. The objective of this study is to assess the prevalence of antibiotic use during Hajj over a period from 2012 to 2017 among French pilgrims and to study the demographic and clinical determinants associated with antibiotic use during this event. We also conducted a systematic review on available evidence for antibiotic consumption in relation to respiratory tract infections (RTIs) during the Hajj.

Methods

Prospective cohort

Participants and study design

Pilgrims from Marseille, France participating in the Hajj from 2012 to 2017 were recruited at a specialized travel agency organizing trips to Mecca. Potential adult participants were invited to participate in the study. They were recruited and followed-up by a medical bilingual (Arabic and French) doctor who traveled with the group. The participants were interviewed using a standardized pre-Hajj questionnaire that collected information on demographic characteristics and medical conditions before departing from France. A post-Hajj questionnaire that collected clinical data and information on the use of antibiotics was completed two days before the pilgrims' return to France.

Influenza-like illness (ILI) was defined in the current study as the presence of subjective fever, sore throat and cough [START_REF] Rashid | Influenza and the Hajj: defining influenza-like illness clinically[END_REF]. Possible pulmonary involvement requiring antibiotic therapy was suspected based on the presence of the following symptoms: productive cough without nasal or throat symptoms; febrile productive cough; dyspnea or febrile dyspnea, according to expert consensus statements [START_REF]Antibiothérapie par voie générale en pratique courante dans les infections respiratoires basses[END_REF][START_REF] Hill | Adult Outpatients With Acute Cough Due to Suspected Pneumonia or Influenza: CHEST Guideline and Expert Panel Report[END_REF]. Streptococcal pharyngitis was suspected based on the presence of fever and sore throat without cough or runny nose [START_REF] Choby | Diagnosis and treatment of streptococcal pharyngitis[END_REF]. The protocol was approved by the Aix-Marseille University institutional review board (July 23 rd , 2013; reference no. 2013-A00961-44). The study was performed according to the good clinical practices recommended by the Declaration of Helsinki and its amendments. All participants provided a written consent.

Statistical analysis

Statistical analysis was conducted using STATA software version 11.1 (Copyright 2009 StataCorp LP, http://www.stata.com). Differences in the proportions were tested by Pearson's chi-square or Fisher's exact tests when appropriate. Unadjusted associations between multiple factors and prevalence of antibiotics use for respiratory symptoms were examined by univariate analysis. The results were presented by percentages and risk ratio (RR) with 95% confidence interval (95%CI).

Results with a p value ≤0.05were considered statistically significant. Only the variables with a prevalence ≥5.0% were considered for statistical analysis. Variables with p values <0.2 in the univariate analysis were included in the multivariate analysis. Log-binomial regression was used to calculate adjusted risk ratios regarding antibiotic consumption [START_REF] Mcnutt | Estimating the relative risk in cohort studies and clinical trials of common outcomes[END_REF].

Review of antibiotic use during the Hajj

Search strategy and selection criteria

This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and MetaAnalyses (PRISMA) guidelines (http://www.prisma-statement. org). The following databases were investigated in an attempt to identify all relevant studies published on: PubMed (http:// www.ncbi.nlm.nih.gov/pubmed) and Google Scholar (http://scholar.google.fr/). The most recent search was conducted on November 12, 2018. The topic search terms used for searching the databases were the following: #1: "Hajj" OR "pilgrims" #2: "antibiotic" OR "antibacterial" OR "antimicrobial" OR "treatment" #3: #1 AND #2 Only articles published in English were included. For inclusion, articles had to fulfil three criteria:

(1) be related to Hajj, (2) be conducted among group of pilgrims independently to the presence of symptoms and (3) report the use of antibiotic. Studies that recruited only sick pilgrims from hospitals or outpatient departments, case reports and reviews were excluded. Reference lists of selected articles were screened to identify studies that might have been missing from the research.

Two researchers (GP and HVT) independently performed the screening of the abstracts. Any discordant result was discussed in a consensus meeting. After screening the abstracts, the full texts of the articles were assessed for eligibility by the same two researchers and selected or rejected for inclusion in the systematic review.

Data collection process

The following data (if available) were extracted from each article: year, countries of origin of pilgrims, study design, sample size of attendees, prevalence of symptoms and ratio of antibiotic use.

Data synthesis and analysis

As a result of the nature of the studies and the heterogeneity in patient populations, a formal metaanalysis was not possible. Therefore, the study results were summarized to describe the main outcomes of interest (i.e. the prevalence of antibiotic use). When possible, percentages not presented in the articles were calculated from the available data.

Results

Cohort survey during the Hajj 2012-2017

Characteristics of study participants

A total of 783 of the 803 pilgrims contacted (97.5%) agreed to participate in our study and completed the questionnaire required before the trip. Of them, 754 (96.3%) also responded to the post-Hajj questionnaire. 328 pilgrims were male (41.9%) with a gender ratio of 1:1.4. The median age was 62 years of age (interquartile = (54-68), min = 21, max = 96 years). Hypertension (28.0%) and diabetes (29.4 %) were the most common comorbidities (Table 1).

Clinical features and antibiotic intake during the Hajj

Figure 1 shows the prevalence of respiratory symptoms and antibiotic use among pilgrims during the Hajj. Most frequent symptoms were cough (77.8%, 587/754), rhinitis (57.3%, 432/754) and sore throat (65.1%, 491/754). 26.1% (197/754) of pilgrims had ILI and 26.3% (198/754) had a possible pulmonary involvement. Only 2 pilgrims presented with symptoms of possible streptococcal pharyngitis and both used antibiotics. At total, 3 patients were hospitalized. Two days prior to leave Saudi Arabia, 46.6% (351/754) of pilgrims were still symptomatic. A total of 47.6% (359/754) of pilgrims took antibiotics during their pilgrimage, representing 55.6% of ill pilgrims. Beta-lactams were the most commonly used antibiotic (35.0%), followed by macrolides (11.4%), cephalosporines (2.3%), quinolones (1.5%) and sulfonamides (0.1%).

Determinants associated with antibiotic consumption during the Hajj and impact of antibiotics on respiratory symptoms

Table 2 shows results of univariate and multivariate analysis about determinants associated with antibiotic consumption. Pilgrims suffering from productive cough or fever were three times and twice as likely, respectively, to have used antibiotics than others (aRR = 2.97, 95%CI [2.28-3.88], p<0.0001 and aRR = 2.01, 95%CI [1.53-2.63], p<0.0001 respectively). Dry cough, sore throat and voice failure were also associated with increased antibiotic use. Of note, antibiotic intake was not significantly increased in individuals with a possible pulmonary involvement compared to others (18.7% versus 28.9% respectively) (Table 2).

With regard to the persistence of respiratory symptoms post-Hajj, no socio-demographic factors and no chronic conditions was associated. Antibiotic intake was associated with increased frequency of symptom persistence (aRR = 1.31, 95%CI [1.04-1.66], p=0.02) (Table 3). This increase was only observed in pilgrims with symptoms of LRTI (RR = 1.86, 95%CI [1.32-2.61], p = 0.0002) but not in those with symptoms of URTI (RR = 0.93, 95%CI [0.75-1.15], p = 0.50) (data not shown).

Review on antibiotic consumption at the Hajj

through the manual search. After screening the titles and abstracts, 14 articles were eventually retained for full-text assessment. All 14 articles were included in the qualitative synthesis of the systematic review (supplementary figure 1). Studies were conducted during the Hajj seasons from 1999 to 2016 and included a total of 7774 pilgrims originating from various countries, including Pakistan, Malaysia, Ireland, US, Singapore, UK, Iran, Australia and India. Most studies were conducted based on a prospective cohort follow-up design and some used a cross-sectional survey design. RTIs were common among pilgrims and hospitalization rates were low. The use of antibiotic for RTIs during the Hajj varied by nationality, from 7% in a Singaporean survey in 2001 to 58.5% among Iranian pilgrims in 2012. In 9 studies out of 14, the antibiotic consumption prevalence was >30% [START_REF] Azeem | Knowledge, Attitude and Practice (KAP) Survey Concerning Antimicrobial Use among Australian Hajj Pilgrims[END_REF][START_REF] Qureshi | The incidence of vaccine preventable influenza-like illness and medication use among Pakistani pilgrims to the Haj in Saudi Arabia[END_REF][START_REF] Ali | An Analysis of the Pattern of Travel-Related Morbidity Reported by Hajj Pilgrims: An Irish Perspective[END_REF][START_REF] Hashim | The prevalence and preventive measures of the respiratory illness among Malaysian pilgrims in 2013 Hajj season[END_REF][START_REF] Dull | Neisseria meningitidis serogroup W-135 carriage among US travelers to the 2001 Hajj[END_REF][START_REF] Wilder-Smith | Acquisition of W135 meningococcal carriage in Hajj pilgrims and transmission to household contacts: prospective study[END_REF][START_REF] Wilder-Smith | Meningococcal carriage in Umra pilgrims returning from Saudi Arabia[END_REF][START_REF] Wilder-Smith | Absence of Neisseria meningitidis W-135 electrophoretic Type 37 during the Hajj, 2002[END_REF][START_REF] El Bashir | Meningococcal W135 carriage; enhanced surveillance amongst east London Muslim pilgrims and their household contacts before and after attending the 2002 Hajj[END_REF][START_REF] Alborzi | Meningococcal carrier rate before and after hajj pilgrimage: effect of single dose ciprofloxacin on carriage[END_REF][START_REF] Metanat | Pharyngeal carriage rate of Neisseria meningitidis before and after the Hajj pilgrimage[END_REF][START_REF] Memish | Mass gathering and globalization of respiratory pathogens during the 2013 Hajj[END_REF][START_REF] Azeem | Surveillance of Australian Hajj pilgrims for carriage of potentially pathogenic bacteria: Data from two pilot studies[END_REF][START_REF] Ganaie | Impact of Hajj on the S. pneumoniae carriage among Indian pilgrims during 2016 -a longitudinal molecular surveillance study[END_REF] (Table 4).

Discussion

Our study confirms that RTIs are common during the Hajj with a high proportion of antibiotic use of 47.6%. The multivariate analysis showed that upper respiratory tract infection (URTI) symptoms (dry cough, sore throat and voice failure), productive cough and fever were independent factors associated with increased antibiotic use. In this study, only 26.3% of pilgrims reported clinical symptoms, suggesting a lower respiratory tract infection (LRTI) that may require antibiotic use according to the French recommendations [START_REF]Antibiothérapie par voie générale en pratique courante dans les infections respiratoires basses[END_REF]. The antibiotic consumption, however, was lower in patients with a possible pulmonary involvement compared to others presenting with symptoms of URTI. According to the French recommendations [START_REF]Antibiothérapie par voie générale en pratique courante dans les infections respiratoires basses[END_REF], only 142/359 (39.6%) of pilgrims who have used an antibiotic had an indication for their use. By contrast, 57/198 (28.8%) pilgrims with a possible LRTI did not receive antibiotic although they had an indication for them. Furthermore, pilgrims who took antibiotics during their stay were significantly more likely to present with persisting symptoms of LRTI, post-Hajj. A possible explanation for this finding is that the sicker patients were more likely to take antibiotics and also more likely to have persistent symptoms.

Alternatively, antibiotics may have altered either the underlying flora or the immune responses in ways that impeded recovery.

The high prevalence of antibiotic use at the Hajj is not observed among French pilgrims only. Our review shows that a high proportion of pilgrims of different nationality used antibiotics during their pilgrimage because of RTIs. In a survey conducted among 1162 pilgrims from 13 different countries in 2013, at the Jeddah airport, 62% ILI was observed after the Hajj and 45.5% received antibiotic [START_REF] Memish | Mass gathering and globalization of respiratory pathogens during the 2013 Hajj[END_REF]. Most RTI cases during the Hajj are URTIs, while pneumonia is uncommon among pilgrims [START_REF] Qureshi | The incidence of vaccine preventable influenza-like illness and medication use among Pakistani pilgrims to the Haj in Saudi Arabia[END_REF][START_REF] Ali | An Analysis of the Pattern of Travel-Related Morbidity Reported by Hajj Pilgrims: An Irish Perspective[END_REF][START_REF] Hashim | The prevalence and preventive measures of the respiratory illness among Malaysian pilgrims in 2013 Hajj season[END_REF][START_REF] Mustafa | A case-control study of influenza vaccine effectiveness among Malaysian pilgrims attending the Haj in Saudi Arabia[END_REF]. Most URTIs are due to viruses with no formal need for antibiotic treatment [START_REF] Cars | Antibiotic use and bacterial complications following upper respiratory tract infections: a population-based study[END_REF].

Antibiotic resistance of bacteria in Saudi Arabia has recently emerged for several reasons, including self-prescribing of antibiotics by patients, irrational or over-prescription by medical staffs, subtherapeutic doses of antimicrobial agents and poor case management by unsuitable combinations and, non-compliance with prescribed treatments by patients. In one study conducted at one hospital in Jeddah, 59.3% Klebsiella pneumoniae respiratory isolates were resistant to ampicillin and piperacillin [START_REF] Alam | A surveillance study on the prevalence and antimicrobial resistance pattern among different groups of bacteria isolated from Western province of Saudi Arabia[END_REF]. In another study, conducted at one hospital in Taif, 30.5% gram-negative bacteria isolates had an extended spectrum β-lactamases phenotype [START_REF] Ghonaim | Risk factors and molecular features of extended-spectrum beta-lactamase producing bacteria at southwest of Saudi Arabia[END_REF]. Recent publications have shown that antibiotic-resistant bacteria acquisition at the Hajj is frequent [START_REF] Leangapichart | Emergence of drug resistant bacteria at the Hajj: A systematic review[END_REF]. Among French pilgrim cohorts sampled in 2013 and 2014 a significant acquisition of extended spectrum β-lactamases-, carpabenemase-producing bacteria or mcr-1-positive isolates was reported [START_REF] Leangapichart | Risk factors for acquisition of CTX-M genes in pilgrims during Hajj 2013 and 2014[END_REF][START_REF] Leangapichart | Acquisition of extended-spectrum b-lactamases by Escherichia coli and Klebsiella pneumoniae in gut microbiota of pilgrims during the hajj pilgrimage of 2013[END_REF][START_REF] Leangapichart | Acquisition of a high diversity of bacteria during the Hajj pilgrimage, including Acinetobacter baumannii with blaOXA-72 and Escherichia coli with blaNDM-5 carbapenemase genes[END_REF][START_REF] Leangapichart | Acquisition of mcr-1 plasmid-mediated colistin resistance in Escherichia coli and Klebsiella pneumoniae during Hajj 2013 and 2014[END_REF]. It is therefore likely that antibiotics used by French pilgrims in the present study, might have been at least partially ineffective in treating LRTI symptoms.

The assessment of the bacterial origin of RTI in pilgrims would necessitate taking a sputum sample before starting treatment for identification and sensitivity testing, since antibiotic intake prior to pneumonia diagnosis can impair the detection of the causative agent [START_REF] Falguera | Etiology and outcome of communityacquired pneumonia in patients with diabetes mellitus[END_REF]. However, most of ill pilgrims are seen in outpatient clinics or by medical missions. In this context, paraclinical diagnosis such as radiology and the culture of respiratory pathogens is difficult and the prescription of antibiotics is often based on the clinical evaluation of the patient. Therefore, antibiotic prescription should be restricted to patients presenting with symptoms of LRTI. Access to antibiotic without prescription is another cause of high prevalence of antibiotic use during the Hajj. A recent knowledge, attitude and practice survey showed that 66.6% pilgrims accessed antibiotics without prescription through a pharmacist. Over 87% of them used non-prescribed antibiotics. A proportion of 79.2% used multiple sources to access antibiotics. Only 12.7% of respondents indicated that in the event of illness, they would visit a clinic and only take the medications prescribed by a doctor. A proportion of 26.5% used antibiotics prescribed to them by a doctor for a previous illness and 10% antibiotics prescribed by doctors for their relatives [START_REF] Yezli | Knowledge, attitude and practice (KAP) survey regarding antibiotic use among pilgrims attending the 2015 Hajj mass gathering[END_REF].

In a study conducted during the Grand Magal de Touba, in Senegal, in 2017, 41.8% pilgrims reported respiratory symptoms and only 2.7% received antibiotics [START_REF] Hoang | Respiratory and gastrointestinal infections at the 2017 Grand Magal de Touba, Senegal: A prospective cohort survey[END_REF]. We are not aware about studies documenting the antibiotic intake among participant to other mass gatherings. Such studies would be of interest.

Our study was based on questionnaires and has some limitations including notably the lack of data about clinical examination of participants, the lack of radiological and microbiological documentation of RTIs and the lack of duration of antibiotic treatment. Also we did not differentiated self-prescription of antibiotics and antibiotics prescription by the accompanying doctor.

Conclusion necessary to follow the recommendations for antibiotic use based on clinical symptoms in pilgrims

in order to raise rational consumption of antibiotics during Hajj. A pilgrim education strategy on Hajj-related pathologies and indications of antibiotics and the promotion of influenza and pneumococcal vaccination is needed. In particular, it is important to organize information sessions before travel to Mecca, or to use documents (flyers) or information online. In addition, it is important to control the delivery of antibiotics in the countries of origin of pilgrims and to enforce the legislations of KSA government [START_REF] Yezli | Knowledge, attitude and practice (KAP) survey regarding antibiotic use among pilgrims attending the 2015 Hajj mass gathering[END_REF]. Rapid identification tests for respiratory pathogens could help medical staff in charge of pilgrims to rationalize their prescriptions for antibiotics.

Figure 1 :

 1 Figure 1: Antibiotic consumption according to respiratory tract infections during the Hajj 2012-2017 (N=783)

Table 2 :

 2 Associated factor to antibiotic consumption during the Hajj (N=754)

	Variables

Table 3 :

 3 Associated factors to persistence of respiratory symptoms at return from the Hajj (uni-and multi-variate analysis (N = 646 ill pilgrims)

				Persistence of respiratory symptoms
	Variables			RR	aRR
			n (%)	P-value	P-value
				[95%CI]	[95%CI]
	Socio-demographic characteristics
	Gender	Male	148 (46.7)	1.01
				0.92
		Female	202 (46.3)	[0.86-1.18]
	Age*	≥ 60 years	214 (56.6)	1.10
				0.18
		<60 years	136 (51.3)	[0.95-1.28]
	Comorbidities
	Diabetes mellitus*	Yes	95 (52.2)	0.95
				0.51
		No	254 (55.1)	[0.81-1.14]
	Hypertension*	Yes	114 (58.8)	1.12
				0.13
		No	235 (52.3)	[0.97-1.30]
	Chronic respiratory	Yes	41 (59.4)	1.11
				0.36
	disease*	No	308 (53.7)	[0.90-1.37]
	Chronic heart	Yes	29 (58.0)	1.07
				0.58
	disease*	No	320 (54.0)	[0.84-1.38]
	Antibiotic consumption
	Antibiotic intake	Yes	209 (58.4)	1.18	1.31
				0.02	0.02
		No	142 (49.3)	[1.02-1.37]	[1.04-1.66]

Table 4 :

 4 Review of literature on antibiotic consumption during the Hajj

	Pilgrim
			Number of		Prevalence of
	age	Study design		Prevalence of respiratory symptoms		Reference
			pilgrims		antibiotic intake
	year
	2013	Cross-sectional study conducted among	1162	Not documented	34.9%. The reason for	14
		Australian Hajj pilgrims in Mina and Mecca,			antibiotic use was: RTIs
		Saudi Arabia			in 83.9% cases
	1999	Prospective cohort study conducted among	2070	ILI (sore throat and cough or temperature	26.6%	19
		Pakistani Hajj pilgrims enrolled in Pakistan		≥ 38°C) 47.9%
		before the Hajj based on identification		URTI (cough or sore throat or rhinitis or
		numbers attributed by the Pakistani		myalgia or headache) 72.2%
		government		Hospitalization 0.3%
	2008	Prospective cohort study conducted among	167	Sore throat or cough or ILI 79% per-Hajj	31%	20
		Irish Hajj pilgrims recruited at a travel clinic.		and 15% post-Hajj
				Hospitalization 1.2% with 1 case of
				pneumonia and 1 case of tonsillitis.

ILI: influenza like illness, URTI: upper respiratory tract infection, RTI: respiratory tract infection

Table 2: Associated factor to antibiotic consumption during the Hajj (N=754)

Table 3: Associated factors to persistence of respiratory symptoms at return from the Hajj (uni-and multi-variate analysis (N = 646 ill pilgrims)

Funding

This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the National Research Agency under the program « Investissements d'avenir », reference ANR-10-IAHU-03, the Région Provence Alpes Côte d'Azur and European funding FEDER PRIMI.

Conflict of Interest

Van-Thuan Hoang, Thi-Thu-Thuy Nguyen, Khadidja Belhouchat, Mohammed Meftah, Doudou