Javouhey Belot

Sylvie Pons

Karen Brengel-Pesce

Yves Gillet

Etienne Alexandre Boisselier

Philippe Rebaud

Romain Basmaci

Nina Droz

PhD Sophie Trouillet-Assant

Sébastien Viel

Antoine Ouziel

Lucille Boisselier

Alexandre Belot

Karen Brengel- Pesce

Etienne Javouhey

Marine Mommert

Audrey Guichard

François Bartolo

Laurence Generenaz

Alexandre Pachot

Claire Capella

Laure Hees

Ellia Mezgueldi

Chadia Toumi

Coralie Bouchiat-Sarabi

Jean-Sebastien Casalegno

Aurelie Portefaix

Romain Deshayes De Cambronne

Magali Perret

Type I interferon in children with viral or bacterial infections

Keywords: infection, viral, febrile children, emergency, bacterial infection Interferon-alpha Interferon-α, febrile children, viral infection, bacterial infection, emergency department

Background: Fever is one of the leading cause of consultation in pediatric emergency department (PED) under the age of 3 years old. Distinguishing between bacterial and viral infections etiologies in febrile patients remains challenging. We hypothesized that specific host biomarkers for viral infections, like type I-interferon (IFN), could help clinicians decisions and limit antibiotic overuse.

Methods

: Paxgene tubes and serum were collected from febrile children (n=101) aged from 7 days to 36 months, attending PED in France with proven viral or bacterial infections. We have assessed the performance of IFN signature which is based on quantification of expression of IFN-stimulated genes using Nanostring® technology and plasma IFN-α quantified by digital ELISA technology (Quanterix ®).

Results: Serum levels of IFN-α were below the quantification threshold (30fg/mL) for 2%

(1/46) of children with proven viral infections and for 71% (39/55) of children with bacterial infections (p<0.001). IFN-α levels and IFN score were significantly higher in viral compared to bacterial infection (p<0.001). We noticed a strong correlation between serum ). Both serum level IFN-α and IFN score robustly discriminated (Area Under the Curve >0.91 for both) between viral and bacterial infection in febrile children, compared to C-reactive protein (0.83).

Conclusions:

This study revealed for the first time, that IFN-α is increased in blood of febrile infants with viral infections. The discriminative performance of IFN-α femtomolar concentrations as well as blood transcriptional signatures could show a diagnostic benefit and potentially limit antibiotic overuse.

Clinical Trials Registration clinicaltrials.gov (NCT03163628).

INTRODUCTION

Type I interferons (IFNs) are a group of cytokines that are involved in the innate antiviral response. A chronic exposure to these cytokines is also linked to the pathophysiology of a group of autoimmune diseases called type I interferonopathies [START_REF] Psarras | Type I interferon-mediated autoimmune diseases: pathogenesis, diagnosis and targeted therapy[END_REF][START_REF] Mcnab | Type I interferons in infectious disease[END_REF]. As it is secreted at very low levels (femtomolar) during disease course, detection of type I IFN in patients remains challenging and led several groups to propose an alternative strategy for the monitoring of this group of cytokines. Based on the quantification of expression of IFN-stimulated genes (ISGs), blood transcriptional signatures (IFN signature) provide an indirect estimate of the exposure of cells to type I IFN and are currently proposed for the screening of autoimmune diseases [START_REF] Rice | Assessment of Type I Interferon Signaling in Pediatric Inflammatory Disease[END_REF].

In addition, the recent development of digital ELISA (Single molecular array, Simoa) allows a very sensitive measurement of type I IFN molecules in the sera of patients.

In children, diagnosis of viral versus invasive bacterial infection is challenging since the main symptoms at onset are often similar and restricted to fever. Misdiagnosis is responsible for inappropriate antibiotic prescription contributing to the emergence of multi-drug resistant bacteria. Thus, we hypothesized that type I IFN, the key cytokine of antiviral response, may represent a new early biomarker of viral infection which could eventually help clinicians limit antibiotic overuse. The aim of this proof-of-concept study was to assess the performance of plasma IFN-α and IFN score in distinguishing documented viral and bacterial infections in a prospective cohort of children attending pediatric emergency departments (PED) for fever.

METHODS

Participants

This is an ancillary study of the prospective multicenter protocol ANTOINE (NCT03163628detailed in supplementary material and Fig- S1). The ANTOINE study was registered to the French National Data Protection Agency under the number 17-168 and has been approved by an ethics committee for biomedical research in May 2017 (Comité de Protection des Personnes Sud Méditerranée II) under the number 217-R18. For each participant, written informed consent was obtained from parents or legal guardians for the participation of the febrile children in the ANTOINE study. An additional specific written consent was required for their inclusion in this present ancillary study. Febrile children aged from 7 days to 36 months attending PED for a suspicion of infection were recruited prospectively in three different hospitals based in Lyon, Villefranche sur Saone and Colombes in France. Inclusion criteria were fever for more than 6 hours (temperature ≥38°C between 7 days and 3 months old and ≥38.5°C between 3 months and 36 months old) for which the physician prescribed venipuncture for suspected severe bacterial infection before any antibiotic treatment. For this study, Paxgene® tubes and serum were collected together with clinical blood tests.

Concomitantly, blood samples from healthy volunteers (HV, n=10) were obtained from the national blood service (Etablissement Français du Sang, Lyon, France, details in supplementary material). In addition, a pediatric disease control population (n=9) with no infection nor characterized type I Interferonopathies was also selected among patients attending consultations at the National Referee Centre for Rheumatic and AutoImmune and Systemic diseases in childrEn (details in supplementary material and patients characteristics detailed available in supplementary Table1).

Diagnostic process

Clinical Chemistry

C o n f i d e n t i a l

The ANTOINE study was designed to evaluate the performance of several biomarkers in

IFN-α and IFN-γ assay

Serum IFN-α concentrations (in fg/ml) were determined by single molecule array (Simoa) using a commercial kit for IFN-α quantification (Quanterix™, Lexington, MA, USA). The assay is based on a 3-step protocol using an HD-1 Analyzer (Quanterix; (4) see IFN score assessment RNA was extracted from whole blood contained in Paxgene® tubes (Kit PreAnalytix, Qiagen©, SW) and quantified by spectrophotometry assay (Nanodrop 2000, Thermo Scientific™, MA, USA). RNA integrity was then evaluated by Agilent RNA microarray (Agilent Technologies©, Santa Clara, CA, USA). The mRNA quantification of 6 ISGs (IFI27, IFI44L, IFIT1, ISG15, RSAD2, SIGLEC1) and 3 housekeeping genes (ACTB, HPRT1, POLR2A), was performed using nanostring technology (Nanostring Technologies©, WA, USA). Data standardization was obtained using the geometric average of internal control and housekeeping genes count number. Interferon score was calculated as previously described [START_REF] Pescarmona | Comparison of RT-qPCR and Nanostring in the measurement of blood interferon response for the diagnosis of type I interferonopathies[END_REF]

Statistical analysis

Non-parametric Mann-Whitney tests and Spearman's correlation were calculated for all parameters using R software V3.6.1. A p-value <0.05 was considered statistically significant.

RESULTS

Serum levels of IFN-α were below the quantification threshold (30fg/mL) for 2% (1/46) of children with proven viral infections and for 71% (39/55) of children with bacterial infections (p<0.001). Median [IQR] IFN-α levels were significantly higher in viral (7856 [3096-62305] fg/mL) compared to bacterial infections (406 [68-3708] fg/mL, p<0.001; Fig1-A). Similarly, the IFN score was increased 30-fold in viral infections compared to bacterial ones (p<0.001; Fig1-B). Interestingly, no significant difference was observed between febrile children with bacterial infections and healthy volunteers or control pediatric population (Fig1-AB). There was a very strong positive correlation between serum IFN-α concentrations and the IFN score In addition, knowing that ISG expression may also be driven by Type II interferon, we then evaluated whether serum IFN-γ concentrations could be correlated with the IFN score. No relation was observed between ISG expression and Type II interferon circulating levels (rspearman 0.18 [-0.054;0.407]; Fig1-F). Of note, no significant difference was observed between IFN-γ serum levels of children with viral infections compared to those with bacterial ones (Fig1-E).

Clinical Chemistry

C o n f i d e n t i a l

DISCUSSION

Biomarkers have become key tools during the clinical decision-making process for clinicians dealing with febrile children. In PED, CRP, procalcitonin (PCT), and white blood cell count are probably the most used markers despite their poor performance in guiding antibiotic prescription in such clinical settings [START_REF] Van Der Does | Procalcitonin-guided antibiotic therapy in patients with fever in a general emergency department population: a multicentre non-inferiority randomized clinical trial (HiTEMP study)[END_REF]. In combination with a marker specific for bacterial infections, a specific biomarker for viral infections could improve the management of febrile children. Recent clinical studies have shown that the combination of CRP or PCT with Myxovirus resistance protein 1 (MxA) coded by the ISG mx1, strongly improved both clinical sensitivity and specificity for differentiating infectious etiology [START_REF] Self | Diagnostic Accuracy of FebriDx: A Rapid Test to Detect Immune Responses to Viral and Bacterial Upper Respiratory Infections[END_REF][START_REF] Shirley | A Rapid Diagnostic Test for Differentiating Bacterial and Viral Aetiologies in Acute Respiratory Infections[END_REF]. Nevertheless, MxA is an intracellular protein which prevents its dosage in serum or plasma, the method currently used for dosage of CRP or PCT in routine labs [START_REF] Zav'yalov | Interferon-Inducible Myxovirus Resistance Proteins: Potential Biomarkers for Differentiating Viral from Bacterial Infections[END_REF]. Here, we have assessed by digital ELISA the performance provided by the measurement of protein IFN-α itself, which is secreted by immune cells after recognition of viruses and detectable in serum or plasma. This preliminary study showed for the first time that IFN-α, at the protein level, is increased in blood of febrile infants with viral infections. The performance of this promising biomarker needs to be confirmed in a larger cohort of febrile children with suspected and proven infections.

Moreover, the biomarkers performance reported in this study have been evaluated in the ANTOINE clinical trial performed in «real-life» condition. In our cohort, only 15% of patients have a definite infection based on the routine microbiology results and the clinical parameters. This is in line with previous reports showing that laboratory tests failed to identify a causative pathogen in over half of children with severe illness or fever leaving an important place for biomarkers to improve the management of febrile children [START_REF] Bleeker-Rovers | A prospective multicenter study on fever of unknown origin: the yield of a structured diagnostic protocol[END_REF][START_REF] Martinón-Torres | Life-threatening infections in children in Europe (the EUCLIDS Project): a prospective cohort study[END_REF]. In our study, the microbiological investigation was not exhaustive for each patient, explaining probably the low prevalence of rhinoviruses in our cohort. Consequently, viral co-infection was not investigated/reported in our cohort. This could explain the high level of interferon alpha observed for some patients with a documented bacterial infection.

The alternative to IFN-α protein quantification, represented by an IFN score measurement, was also evaluated in this study. Results suggest that this ISG-based signature has the same diagnostic performance than IFN-α protein quantification. Fast track RNA analyses using Nanostring® or FilmArray® platforms could thus be helpful to implement this marker in routine use. Of note, the circulating IFN-α concentrations as well as the IFN scores reported herein in case of viral infections were in the same order of magnitude than those observed in cases of autoimmune disease such as systemic lupus erythematosus (SLE) [START_REF] Oke | High levels of circulating interferons type I, type II and type III associate with distinct clinical features of active systemic lupus erythematosus[END_REF]. Moreover,

Clinical Chemistry

C o n f i d e n t i a l

several recent studies suggest that IFN-α could be a useful marker to monitor interferonopathie disease activity, to identify patients with high risk of relapse, and to select the best candidates for anti-IFNα treatment [START_REF] Mathian | Ultrasensitive serum interferon-α quantification during SLE remission identifies patients at risk for relapse[END_REF][START_REF] Mathian | Monitoring Disease Activity in Systemic Lupus Erythematosus With Single-Molecule Array Digital Enzyme-Linked Immunosorbent Assay Quantification of Serum Interferon-α[END_REF][START_REF] Melki | Anti-MDA5 juvenile idiopathic inflammatory myopathy: a specific subgroup defined by differentially enhanced interferon-α signalling[END_REF]. Our results suggest that viral infections could interfere and possibly be responsible of false positive diagnoses when the IFN score or IFN-α protein is used for the screening and the monitoring of autoimmune diseases.

Of note, recent published data demonstrated that in specific clinical diseases, such as Mycobacterium tuberculosis infections, ISG expression was not mediated through plasma type I IFN [START_REF] Llibre | Plasma Type I IFN Protein Concentrations in Human Tuberculosis[END_REF]. However, in febrile infants, the results herein showed that the expression of ISG was driven by IFN-α and not by IFN-γ.

According to the 68 th World Health Assembly declaration, it is urgent to find effective diagnostic tools to guide optimal antibiotic use [START_REF] Who | Antimicrobial resistance: global report on surveillance[END_REF]. Our study showed that measurement of IFN-α femtomolar concentrations as well as the use of an IFN score could offer new perspectives for improving diagnosis and limiting antibiotic overuse in febrile infants.

«

  real-life » condition. No other blood tests nor clinical investigation has been performed in addition to those requested by the emergency physician caring for febrile children. All patients underwent routine investigations as part of clinical care including blood count and differential, C-reactive protein, blood chemistry, blood, stools and urine cultures, and cerebrospinal fluid analysis where indicated. Chest radiographs were undertaken as clinically indicated. Bacteria culture and viral diagnostics using multiplex PCR were performed by routine microbiological laboratories of each hospital. For patients with suspicion of Respiratory Tract Infections (RTIs), specific PCRs targeting influenza virus or respiratory syncytial virus were performed systematically during seasonal peak. According to the routine microbiology results and the clinical parameters recorded in a numerical clinical record folder (e-CRF), we retained patients with proven viral (n=46) or bacterial (n=55) infections for this study. The infections were proven on the basis of a definite identification of pathogens using routine microbiology testing. Assignment of patients to clinical groups was validated by consensus of an independent adjudication committee (cohort description in Table1).

  (r-spearman [95%CI] 0.85 [0.76;0.91] Fig1-C).

  Analysis of the Area Under the Curve [AUC -95%CI] indicated that both IFN-α serum level (0.930 [0.877;0.983]) and IFN score (0.908 [0.845;0.971]) robustly discriminated viral infections from bacterial ones in febrile children (Fig1-D). To evaluate if IFN-α could improve the diagnosis of febrile children attending PED, we assessed the performance of this biomarker in combination with a clinically validated test that is the C-reactive protein (CRP) quantification. The AUC shown in Figure 1D revealed that the ratio of the concentrations of CRP and IFN-α strongly improves the classification of these patients compared to CRP alone (0.936 [0.888; 0.985] versus 0.829 [0.747;0.910] respectively, Fig1-D).

Acknowledgements

We thank Dr. Jonathan Lopez, Pauline Desormeaux and Isabelle Mosnier (Plateforme de Recherche de transfert en Oncologie des Hospices Civils de Lyon) for his technical assistance on nanostring molecular biology. We thank Véréna Landel for language editing and critical reading of the manuscript.

Supplementary

Methods, available at http://onlinelibrary.wiley.com/doi/10.1002/art.40792/abstract)). IFN-γ concentrations were measured using the simple plex kit IFN-γ assay with the Ella platform (Protein simple©, CA, USA), according to manufacturers' instructions.

Clinical Chemistry

C o n f i d e n t i a l C o n f i d e n t i a l


Frédéric Kratz

José Ragot

Didier Maquin

Antoine Despujols

Detection of bias in measurement using analytical redundancy

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


Fabien Mahaut

Georges Gauthier

Philippe Gouze

Linda Luquot

Dominique Salin

Jérôme Martin
email: martin@fast.u-psud.fr

Rheological characterization of olivine slurries, sheared under CO 2 pressure

Keywords: carbon sequestration, mineral carbonation, slurry injection, well remediation, Bingham plastic

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

I. INTRODUCTION

One of the main concern regarding CO 2 geological sequestration is the risk of leakage through fractures in either the caprock or the plugged injection wells. Fracturing due to mechanical or chemical stresses may be triggered by natural events (e.g. tectonics) but also by the well completion or the CO 2 injection. For a leaky well, the prevailing strategy, inherited from oil industry, is to fill the well with viscous (heavy) slurries and/or concrete. If the leakage issues from the alteration of the cements or the caprock around the well (outside the steel tubing), then cement is locally injected. But although complex and costly, such remediation may not be fully satisfactory. As the injected cement can hardly penetrate deeply in porous media or in small aperture rough fractures, the interface between the sealing cement and the leaky material may remain mechanically weak. Furthermore, albeit portland-type cements are considered as poorly reactive with CO 2 , some recent experiments [START_REF] Bachu | Experimental assessment of brine and/or co2 leakage through well cements at reservoir conditions[END_REF] indicate that they may react when exposed to flowing CO 2 -rich fluid. Accordingly, the use of such cements for sealing issues is questionable. Good alternative candidates could be light slurries (suspensions) composed of magnesium silicate minerals (e.g. olivine or serpentine). Such minerals were investigated by O'Connor et al. [START_REF] O'connor | Carbon dioxide sequestration by direct mineral carbonation with carbonic acid[END_REF] for ex situ carbonation/uptake of CO 2 , as their carbonation, at high temperature and under high CO 2 pressure, enables mineral carbon dioxide sequestration. And as the carbonation of olivine (forsterite M g 2 SiO 4 ) leads to the formation of magnesite (M gCO 3 ), with an increase of the solid phase volume which can reach 88 % [START_REF] Andreani | Experimental study of carbon sequestration reactions controlled by the percolation of co2-rich brine through peridotites[END_REF], O'Connor et al. [START_REF] O'connor | Aqueous mineral carbonation: Mineral availability, pretreatment, reaction parametrics, and process studies[END_REF] suggested that olivine slurries could also be used for in situ preventive reinforcement of wells. Such a technique would present several advantages: olivine and serpentine are present worldwide in greenstone belts and ophiolites, ultramafic rocks grinding is already used in industrial applications (refractory cements, light blasting, abrasives, etc). Moreover, as a candidate for CO 2 sequestration process, the dissolution/carbonation of olivine and serpentine is already well documented [START_REF] Chen | Chemistry of aqueous mineral carbonation for carbon sequestration and explanation of experimental results[END_REF][START_REF] Hänchen | Validation of a population balance model for olivine dissolution[END_REF][START_REF] Krevor | Enhancing serpentine dissolution kinetics for mineral carbon dioxide sequestration[END_REF], and although such a carbonation process may be too expensive for mineral sequestration purposes, it could lead to a rather cheap and efficient process for either preventive well reinforcement or sealing issues. However, the technique is still speculative because of the lack of experimental data.

The present work aims at characterizing the rheometric properties of olivine slurries, and their time evolution. We first present measurements of the apparent viscosity of sheared 2 (at 100 s -1 ) aging olivine slurries. The olivine volume fraction is varied in the range 10 -40 %, the CO 2 pressure in the range 20 -50 bars, and the temperature in the range 60 -110 • C. These tests show that a time laps exists, during which the olivine slurries keep a rather low apparent viscosity which should allow injection. The rheologic properties of the slurries at this early stage are then further characterized by their stress response to different shear rates. The olivine slurry is shown to obey a non-Newtonian Bingham equation. The corresponding yield stress and Bingham viscosity both increase with the olivine volume fraction, in accordance with models found in the literature [START_REF] Wildemuth | Viscosity of suspensions modeled with a sheardependent maximum packing fraction[END_REF][START_REF] Krieger | Rheology of monodisperse latices[END_REF].

II. OLIVINE SLURRIES AND EXPERIMENTAL DEVICES

A. Olivine Suspensions

Olivine suspensions were prepared with Volvic mineral water and olivine particles, of chemical composition M g 1.8 F e 0.2 SiO 4 (with traces of iron sulfate F eSO 4 , diopside CaM gSi 2 O 6 and spinel M gAl 2 O 4 ) provided by Micronizzati MS. However, the initial particle size distribution was rather high (Fig. 1a), with most of the particles in the range 10 -100 µm (D 90 ≃ 100 µm, i.e. 90 % of the particles have a diameter smaller than 100 µm), and their sedimentation rate was too high to conduct rheological characterization, and to expect the suspensions to be injectable. To cope with this problem, the particle size was first decreased to D 90 ≃ 10 µm (Fig. 1b), using air flow micronization in a spiral jet mill, which did not change the chemical composition. With such a size reduction, the typical Stokes velocity was about 130 µm/s for an olivine particle of density 3.36 g/cm 3 , and of typical diameter 5 µm. The slurries prepared at various olivine fractions ϕ, were found to be fluid at ϕ < 30 %, and very pasty at higher concentrations. Therefore we focused our studies on suspensions at ϕ < 30 %, which were easier to handle and which were more likely to be injectable.

B. Rheometer Setup

In order to characterize the evolution of rheological properties, at fixed temperature and CO 2 pressure, we used an Anton Paar MCR501, equipped with a High Pressure (HP) cell. This commercial HP cell enables rheological tests at pressures up to 150 bars and controlled temperatures up to 300 • C, but has some drawbacks.

First, the HP cell has only one geometry (Couette concentric cylinders, of inner and outer radii R i = 12.5 mm and R e = 13.5 mm, over a height h = 36 mm), and the bob has a conical extremity whereas the cup has a plane bottom. As a consequence, the volume between the cup bottom and the bob extremity is submitted to a shear rate significantly lower than the shear rate imposed in the gap of the cell. As a result, both shear-induced diffusion [START_REF] Leighton | The shear-induced migration of particles in concentrated suspensions[END_REF] and sedimentation drive the particles toward the bottom of the cell. The suspension may become non-uniform, leading to erroneous rheological measurements and time characterization. To prevent such non-uniformity, we have used a designed in-house cup, with a conical bottom (Fig. 2). The resulting geometry ensures an almost homogeneous shear rate in the whole volume of sheared suspension, and prevents the migration of particles [START_REF] Deboeuf | Particle pressure in a sheared suspension: A bridge from osmosis to granular dilatancy[END_REF]. The second drawback of the HP cell is due to its magnetic coupling which decreases the accessible range of the rheometric measurements. Whereas the minimum torque is equal to 0.01 µN.m in classical cells, it is increased to 100 µN.m in the HP cell, which corresponds to a shear stress of about 3 P a, and, for a typical shear rate of 100 s -1 , to a minimum measurable viscosity of about 30 mP a.s.

III. RHEOLOGICAL CHARACTERIZATION

A. Evolution of apparent viscosity: Aging of sheared Suspensions

In order to address the aging time scales and the fluidity evolution of the olivine suspensions, we first monitored the apparent viscosity, measured at the constant shear rate γ = 100 s -1 , for different olivine concentrations and thermodynamic conditions.

Note that the value of the shear rate has been selected to ensure an efficient stirring and a measurable viscosity value, for olivine suspensions of typical volume fraction 15 -20 %.

The experimental procedure is the following: A volume of 3 cm 3 of freshly mixed olivine suspension is introduced in the HP cell, at room temperature T r = 20 • C. The cell is closed, connected to carbon dioxide at pressure P CO 2 , bleeded, and connected again. The cell is then isolated and heated up to the set point temperature T , which results in an increase of the pressure in the cell (in the expected ratio T (K)/T r (K), where T (K) is the temperature in Kelvin), whereas the apparent viscosity measured at constant shear rate, is recorded.

A typical record of the apparent viscosity, obtained for an olivine suspension at volume fraction ϕ = 20 %, at γ = 100 s -1 , T = 80 • C, and P CO 2 = 40 bars, is displayed in Figure 3. Three stages may be identified. The first one has a duration of about one hour. It corresponds to the initial onset of a uniform temperature T inside the cell. It is accompanied by a corresponding increase of the CO 2 pressure, and ends with initial plateau values for both the pressure and the measured viscosity. The second stage, of variable duration (about 8 hours in the displayed example), corresponds to an increase of the apparent viscosity, which can be fitted by an exponential growth (dotted blue line). The third stage corresponds to a very noisy decrease of the viscosity measurement, down to unmeasurable values. This unexpected decrease of the viscosity may be enlightened by the comparison of the slurry state in the early second stage (at time t = 2 h), and at the end of the third one (t = 12 h), for the parameters of Figure 3.

At t = 2 h, the slurry is found in a homogeneous state very similar to the initial one, which is a rather fluid state for suspensions at ϕ = 20 %. When the bob is removed from the cell, the fluid slurry flows down, and leaves a thin layer of suspension (left snapshot of Figure 4).

On the opposite, when the cell is opened at t = 12 h, two phases can be identified: A waterlike supernatant liquid phase, and a very thick pasty phase, which sticks to the surfaces of the cup and the bob. When the bob is removed from the cell, the slurry does not flow at all, and stays in a thick solid layer on the bob surface (right snapshot of Figure 4). It is conceivable then that the third stage of the apparent viscosity record corresponds to the fracturing of the thickening slurry, which generates noise in the rheogram, and results in the localization of the shear in a layer of suspending fluid (shear banding effect). However, such an effect may be seen as an artefact of our rheological test, in which a constant shear rate is imposed on a setting slurry, with no limitation of the required stress. In practical applications, the pressure head is fixed (or at least limited), and the phenomena observed during the third stage are likely to be irrelevant: the thickening of the slurry toward a solid state would result in clogging, rather than in an apparent conductivity increase, that one could naively expect from the decrease of the measured viscosity. For these reasons, we focus in the following on the second phase, which is indeed more relevant to address injection issues.

To get some insight into the cause of the thickening, the records of the apparent vis-cosity, under carbon dioxide and under nitrogen are compared in Figure 5. In the absence of CO 2 , no thickening of the olivine slurry is observed, during a 5 h long test: It has been suppressed, or at least delayed. Moreover, the comparison of the suspensions (bottom photographs of Figure 5) reveals that in the suspensions aging under CO 2 pressure, the particles form clusters, which is expected to result in the thickening of the suspensions [START_REF] Quemada | Rheology of concentrated disperse systems and minimum energy dissipation principle[END_REF]. These observations lend credit to the formation of clusters of olivine particles, stuck by the olivine dissolution-carbonation reaction:

M g 2 SiO 4 + 2CO 2 → 2M gCO 3 + SiO 2 .
(

) 1
Such a reaction is substantiated by local chemical analysis using a Scanning Electron Microscope (SEM) of the solid phase after 7 h and 13 h of aging process (Figure 6). First, the size observed for the initial particles, and for the 7 h and 13 h old suspensions, is smaller and smaller, which supports some acid dissolution of the olivine. Second, the products of the olivine dissolution-carbonation reaction, namely phases rich in carbonate, iron oxyde and silicon (which could be either silica or amorphous silicon) are present after aging under CO 2 as observed by Andreani et al. [START_REF] Andreani | Experimental study of carbon sequestration reactions controlled by the percolation of co2-rich brine through peridotites[END_REF].

To characterize the kinetics of the rheological evolution of the olivine slurries, the influence of the different parameters on the evolution of the apparent viscosity has been studied and quantified by the estimation of the characteristic time τ of the exponential increase of viscosity during the second stage. of the olivine, which obeys an Arrhenius law r ∝ exp(-E a /RT ). Indeed, the semi-log plot of the variations of τ ∝ 1/r with 1/T (K) (inset of the bottom Figure 7) is rather linear.

Moreover, the corresponding slope E a /R gives E a = 66.2 kJ/mol, comparable with the value E a = 66.5 kJ/mol obtained by Jonckbloedt [START_REF] Jonckbloedt | Olivine dissolution in sulphuric acid at elevated temperaturesimplications for the olivine process, an alternative waste acid neutralizing process[END_REF] in acidic solutions. We note however that such a good agreement may be rather fortuitous, as E a has been reported to vary from 38 to 126 kJ/mol, depending on the experimental conditions [START_REF] Hänchen | Dissolution kinetics of fosteritic olivine at 90-150 c including effects of the presence of co2[END_REF]. The different values of τ , obtained for CO 2 pressures fixed between 20 bars and 52 bars, at ϕ = 20 %, γ = 100 s -1 , T = 90 • C, are displayed in Figure 8. Although the experiments display some variability, a decrease of P CO 2 is clearly associated with a delay of the apparent viscosity increase. This trend is illustrated in the graph τ (P CO 2 ): The characteristic time is decreased by a factor of about five when the CO 2 pressure is increased from 20 bars to 50 bars. The data are also displayed in log-log scale (inset of the bottom Figure 8) to test a power law behavior. The best fit has an exponent of 2.15, but it is unclear if the slope remains constant or increases from ∼ 2 at low P CO 2 to ∼ 0.5 at high P CO 2 . Actually, the solution in our experiments is expected to be mildly acid (mineral water at pH = 7, under CO 2 pressure), and the kinetics of the dissolution of the olivine, in this range of experimental conditions, may be rather complex: The order of the pH dependence may change around pH = 5 [START_REF] Hänchen | Dissolution kinetics of fosteritic olivine at 90-150 c including effects of the presence of co2[END_REF] or pH = 7 [START_REF] Wogelius | Olivine dissolution kinetics at near-surface conditions[END_REF],

and depends also on the concentrations of the other ions [START_REF] Wogelius | Olivine dissolution kinetics at near-surface conditions[END_REF][START_REF] Pokrovsky | Kinetics and mechanism of forsterite dissolution at 25 c and ph from 1 to 12[END_REF]. Besides, we note that the smaller estimated values of the characteristic times (τ ∼ 1.5 h) are the lower values accessible in our experiments: They correspond to a continuous transition between the onset of the experimental conditions of temperature and pressure (first stage) and the divergence of the measured apparent viscosity (third stage). The measurement of shorter times τ , expected for higher pressures P CO 2 , are thus beyond the time resolution of our experimental protocol.

Thence, our data on the effect of P CO 2 on the rheological aging of a flowing slurry remain partly inconclusive. Further understanding would require to enlighten our data, obtained under controlled flow conditions, with data obtained under controlled chemical conditions (with the monitoring of the pH and chemical concentrations).

To complete the aging issue, the apparent viscosities recorded for different concentrations of olivine (15 % < ϕ < 30 %) are displayed in Figure 9. A significative delay of the slurry evolution is observed when the concentration is decreased down to ϕ = 15 %. Conversely, τ vanishes for initial concentrations of olivine higher than 30 % (bottom graph of Figure 9).

In this range (30 % < ϕ < 40 %), the initial slurries were pasty, and it was actually difficult to fill the cell, because they did not flow out of the vessel. This suggests that these slurries have a yield stress, under which they would not flow. This property cannot be evaluated with an apparent viscosity record, and requires a more complex rheological characterization, such as stress-strain curves discussed in the following.

two aging times t = 2 h and t = 4 h. As expected, the initial and final values at γ = 100 s -1 are comparable: There is no hysteresis. Moreover, the response is well described with a Bingham-plastic model of the form:

Σ = Σ B + µ B γ, ( 2
)
where Σ B is a yield stress and µ B is the Bingham viscosity.

These quantities, measured for a volume fraction between ϕ = 10 % and ϕ = 30 %, are displayed in Figure 11. The yield stress Σ B increases with ϕ, from 1 P a to 20 P a, which is in accordance with the above mentioned observation, that the pasty initial slurry does not flow from the vessel by its own weight at ϕ > 30 %. Such an order of magnitude (20 P a at ϕ = 30 %) is also typical of slurries containing fine or ultrafine particles (particle size under 5 µm) [START_REF] He | Slurry rheology in wet ultrafine grinding of industrial minerals: a review[END_REF][START_REF] Chun | Stabilization and control of rheological properties of Fe 2 O 3 /Al(OH) 3 -rich colloidal slurries under high ionic strength and ph[END_REF][START_REF] Chun | Effect of particle size distribution on slurry rheology: Nuclear waste simulant slurries[END_REF], which are known to promote a yield stress. We note that in this kind of slurries, the rheology is not controlled solely by the hydrodynamic interactions, but depends also on Brownian interactions: Van der Walls, electrostatic... Thence, the rheology (and especially the yield stress) is expected to vary with the particle size distribution (which evolves with the dissolution/carbonation of the olivine), and with the temperature, pH, chemical composition of the dispersant [START_REF] He | Slurry rheology in wet ultrafine grinding of industrial minerals: a review[END_REF][START_REF] Chun | Stabilization and control of rheological properties of Fe 2 O 3 /Al(OH) 3 -rich colloidal slurries under high ionic strength and ph[END_REF][START_REF] Chun | Effect of particle size distribution on slurry rheology: Nuclear waste simulant slurries[END_REF][START_REF] Zhou | Yield stress and maximum packing fraction of concentrated suspensions[END_REF], which leads to a rather complex slurry rheology, but also provides some monitoring tools.

IV. DISCUSSION

First, our Bingham viscosity measurements can be fitted with a classical Krieger-Dougherty law [START_REF] Krieger | Rheology of monodisperse latices[END_REF] (see Figure 11, bottom),

µ B = µ 0 (1 -ϕ/ϕ max ) -n , (3)
with µ 0 = 3.8 mP a.s , ϕ max = 34.7 % and an exponent n = 1.68, in the range of the values reported in the literature (1.5 [START_REF] Ball | Dynamics of colloidal dispersions[END_REF], 1.82 [START_REF] Krieger | Rheology of monodisperse latices[END_REF], 2 [START_REF] Ovarlez | Local determination of the constitutive law of a dense suspension of noncolloidal particles through magnetic resonance imaging[END_REF]). The values of µ 0 and ϕ max are more surprising. First, µ 0 = 3.8 mP a.s is significantly higher than the value for the suspending water µ water = 0.3 mP a.s at T = 90 • C. Second, the value ϕ max = 34.7 %, which represents the packing fraction of the suspension, is unexpectedly small, compared to typical values 58 % [START_REF] Gauthier | Gravity waves at the interface between miscible fluids and at the top of a settling suspension[END_REF] or 60.5 % [START_REF] Ovarlez | Local determination of the constitutive law of a dense suspension of noncolloidal particles through magnetic resonance imaging[END_REF] for suspensions of spheres [START_REF] Ovarlez | Local determination of the constitutive law of a dense suspension of noncolloidal particles through magnetic resonance imaging[END_REF][START_REF] Gauthier | Gravity waves at the interface between miscible fluids and at the top of a settling suspension[END_REF]. We must stress however that in the present case, the system is more a gel than a suspension, as it has developed a yield stress.

This yield stress compares very nicely to the analytical expression proposed by Wildemuth and Williams [START_REF] Wildemuth | Viscosity of suspensions modeled with a sheardependent maximum packing fraction[END_REF] (see Figure 11, top):

Σ B (P a) = σ 0 ( ϕ/ϕ 0 -1 1 -ϕ/ϕ m ) p , (4)
with σ 0 = 0.22 P a, ϕ 0 = 3.2 %, p = 0.79 and ϕ m = 39.6 % (in line with the low value of ϕ max = 34.7 % found for the viscosity divergence). The so-obtained fitting values indicate that for an olivine volume fraction of about ϕ 0 = 3.2 %, a yield stress of about σ 0 = 0.22 P a is expected to arise. We note that the "gel" transition value, ϕ 0 = 3.2 %, is significantly higher than the values encountered in colloidal suspensions. Indeed, in the present case, the yield stress is expected to be promoted by the aggregation of the ultrafine (Brownian) particles [START_REF] He | Slurry rheology in wet ultrafine grinding of industrial minerals: a review[END_REF][START_REF] Chun | Effect of particle size distribution on slurry rheology: Nuclear waste simulant slurries[END_REF], which only represent one part of the particles. Moreover, one expects some interplay between their aggregation and the aging conditions: The shear rate at γ = 100 s -1 may promote the aggregation [START_REF] Guery | Irre-versible shear-activated aggregation in non-brownian suspensions[END_REF], whereas the shear stress may delay it though the breaking of the loose aggregates [START_REF] Wildemuth | Viscosity of suspensions modeled with a sheardependent maximum packing fraction[END_REF]. Moreover, the aggregates of ultrafine particles may sinter larger olivine particles which will then contribute to promote the yield stress.

The complexity of the system would require even more characterization in order to describe the complete scenario of the aging of the olivine suspension, during injection. In particular, the dissolution/precipitation reactions taking place in heterogeneous flows in fractures may be rather unexpected [START_REF] Andreani | Experimental study of carbon sequestration reactions controlled by the percolation of co2-rich brine through peridotites[END_REF]. However, the trend plots characterizing the influence of olivine concentration, temperature, and CO2 pressure, are encouraging: A significant time laps (several hours to one day or longer) exists before the thickening of the slurry, which should enable the slurry injection for sealing purposes. We note however that in the explored range of parameters, it is more likely that the stiffening of the slurry is controlled by the olivine dissolution [START_REF] Hänchen | Validation of a population balance model for olivine dissolution[END_REF], rather than by the carbonate precipitation [START_REF] O'connor | Aqueous mineral carbonation: Mineral availability, pretreatment, reaction parametrics, and process studies[END_REF]. In the case of a post injection sealing strategy, monitoring the delay of the olivine carbonation, in the various conditions encountered in the geological sites [START_REF] Wang | The effects of co2-brine rheology on leakage processes in geologic carbon sequestration[END_REF] is still an open challenging issue.

However, with the prospect of a primitive well reinforcement process, the present Bingham characterization of the initial slurry gives the frame of a proper injection strategy [START_REF] Chen | The flow and displacement in porous media of fluids with yield stress[END_REF][START_REF] Yun | Fractal analysis of herschel-bulkley fluid flow in porous media[END_REF][START_REF] Sochi | Modelling the flow of yield-stress fluids in porous media[END_REF].

V. ACKNOWLEDGEMENT

This work was supported by the COLINER project (ANR-08-PCO2-004) funded by the for the yield stress and by Krieger and Dougherty [START_REF] Krieger | Rheology of monodisperse latices[END_REF], for the Bingham viscosity, respectively.

Figure 7

 7 displays the curves obtained at temperatures between 60 • C and 110 • C, at ϕ = 20 %, γ = 100 s -1 , P CO 2 = 40 bars. A strong decrease of τ (from 14 h to 2 h) is observed when the temperature is increased from 60 • C to 80 • C, whereas τ hardly changes (i.e. τ remains at a value close to our time resolution) when the temperature is further increased. It is likely that τ is controlled by the dissolution rate r

FIG. 1 :FIG. 3 :FIG. 6 : 20 FIG. 7 :FIG. 8 :

 1362078 FIG.1: Size distribution and snapshots of the olivine particles, before (left) and after (right) micronization.

22

 22

FIG. 9 :

 9 FIG. 9: Comparison of the apparent viscosities recorded at different olivine concentrations (top) and the corresponding characteristic times τ (bottom), obtained for tests at γ = 100 s -1 , T = 90 • C, P CO 2 = 40 bars.

FIG. 10 :

 10 FIG. 10: Top: Effective viscosity record, at γ = 100 s -1 (black symbols) and response to shear rate steps (red open squares), of an olivine sample, at ϕ = 20 %, T = 90 • C, P CO 2 = 40 bars. The inset displays the shear steps sequence (blue dotted line, right axis) applied at time t = 2 h, and the corresponding stress response (left axis, open red squares and black lines are the recorded values and their measurements, respectively). Bottom: Stress-strain values measured at times t = 2 h and t = 4 h (square and diamond symbols, respectively), compared with the corresponding Bingham plastic curves (straight lines).

FIG. 11 :

 11 FIG. 11: Yield stress (Σ B , top) and Bingham viscosity (µ B , bottom) measured on slurries, at different olivine concentrations, sheared 2 hours at γ = 100 s -1 , T = 90 • C, P CO 2 = 40 bars. The lines fits to the data, using expressions (4) and (3) proposed by Wildemuth and Williams [1],

B. Bingham Plastic Characterization

To address the issue of the non-Newtonian behavior of the olivine slurries, some rheological tests have been carried out, as the response to either steps in shear rate or steps in shear stress. These tests require a time window, during which the system will not evolve significantly. Thence, they have to be applied during the plateau of the second stage (see Figure 3), in slurries aging with a large enough characteristic time τ . Both steps in shear rate or in shear stress have been tested. However in the latter case, the system was difficult to control: The rheometer rotation was stopped at low enough applied stress, whereas a very high shear rate was obtained for stresses above some value. Such a response evidences the existence of a yield stress. We note also that it was associated with important hysteresis, probably due to strong modifications of the slurry state in the absence of stirring. To circumvent this drawback, the non-Newtonian behavior of the olivine slurries was investigated through the response to shear rate steps.

The top panel of Figure 10 displays the apparent viscosity measured on an olivine slurry, at ϕ = 20 %, T = 90 • C, P CO 2 = 40 bars, which was submitted to shear rate steps every 2 hours (apparent viscosity in red symbols), and continuously sheared at γ = 100 s -1 in the mean time (apparent viscosity in black symbols). First at all, the record of the apparent viscosity at γ = 100 s -1 compares very nicely with the one obtained for similar parameters, and without shear steps, displayed in Figure 3, during the first 6 hours. However, at t = 6 h, the response to the third shear steps sequence is followed by a discontinuity of the apparent viscosity, which becomes more noisy thereafter (probably due to the appearance of some shear banding phenomena). And we note that the subsequent shear steps sequences display also some hysteresis (i.e. discontinuity of the apparent viscosity). For the above reasons, we will focus in the following on the response to the first sequence, at t = 2 h, as it does not alter the aging process, and it applies to a "quasi-static" state of the slurry. This shear rate steps sequence consisted in three 100 s long steps at γ = 50, 150, 200 s -1 (blue dotted line, right axis, in the inset of Figure 10).