David Picard
email: david.picard@cnrs-orleans.fr

Laurent Arbaret

Michel Pichavant

Rémi Champallier

Patrick Launeau

Rheology and microstructure of experimentally deformed plagioclase suspensions

Keywords: plagioclase fabrics, magma rheology, experimental deformation, strain partitioning

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

During crystallization, magmas evolve from a viscous suspension, the rheological behaviour of which, mainly controlled by the liquid phase, can be modelled using the Einstein-Roscoe equation [START_REF] Roscoe | The viscosity of suspensions of rigid spheres[END_REF][START_REF] Ishibashi | Viscosity measurements of subliquidus magmas: Alkali olivine basalt from the Higashi-Matsuura district[END_REF], to an interconnected crystalline framework capable of transmitting deviatoric stresses [START_REF] Petford | Rheology of granitic magmas during ascent and emplacement[END_REF] and which exhibits non-Newtonian behaviour [START_REF] Caricchi | Non-Newtonian rheology of crystal-bearing magmas and implications for magma ascent dynamics[END_REF][START_REF] Lavallée | Non-Newtonian law for highly crystalline dome lavas[END_REF][START_REF] Champallier | Experimental investigation of magma rheology at 300MPa: From pure hydrous melt to 75[END_REF]. This rheological evolution is essentially controlled by the crystal fraction and, for a given crystal fraction, by the strain rate [START_REF] Caricchi | Non-Newtonian rheology of crystal-bearing magmas and implications for magma ascent dynamics[END_REF][START_REF] Mueller | The rheology of suspensions of solid particles[END_REF].

However, the morphological characteristics of particles (length, width and aspect ratio) are suspected to have a major influence on the rheology of suspensions [START_REF] Chong | Rheology of concentrated suspensions[END_REF][START_REF] Nicolas | Kinematics in magmatic rocks with special reference to gabbros[END_REF][START_REF] Petford | Which effective viscosity ?[END_REF]. Crystals with different aspect ratios have been considered in analogical and numerical modelling of the microstructural evolution of crystal-melt suspensions [START_REF] Ildefonse | Effect of mechanical interactions on the development of shape preferred orientations : a two dimensional experimental approach[END_REF][START_REF] Saar | Numerical models of the onset of yield strength in crystal-melt suspensions[END_REF]. So far, high temperature and high pressure experimental deformation studies on magma rheology have focused on suspensions made of quasi isometric particles; corundum [START_REF] Champallier | Experimental investigation of magma rheology at 300MPa: From pure hydrous melt to 75[END_REF], quartz [START_REF] Caricchi | Non-Newtonian rheology of crystal-bearing magmas and implications for magma ascent dynamics[END_REF], or natural partially crystallized suspensions [START_REF] Caricchi | Rheological properties of magma from the 1538 eruption of Monte Nuovo (Phlegrean Fields[END_REF][START_REF] Cordonnier | Rheological properties of dome lavas: Case study of Unzen volcano[END_REF] have been investigated. Here, we present the first deformation experiments at high temperatures and pressures of synthetic suspensions of plagioclase, a representative strongly anisometric crystalline particle. With increasing cristallinity, a fundamental modification of the rheological behaviour is demonstrated and correlated with a dramatic evolution of the microstructure of the suspension. Our results stress the importance of the shape of crystals for natural magmatic flows.

EXPERIMENTAL AND CHARACTERIZATION METHODS

Plagioclase-bearing magmatic suspensions with crystal fractions (φ s ) of 0.38 and 0.52 have been synthesized in an internally-heated pressure vessel for 7 days at 300 MPa and 900 and 800°C, respectively. The resulting suspensions consist of homogeneously distributed euhedral plagioclase crystals embedded in a silicic glass containing some gas bubbles, indicating H 2 O-saturated conditions (Fig. 1). Calculated glass H 2 O concentrations [START_REF] Papale | The compositionnal dependence of the saturation surface H 2 O+CO 2 fluids in silicate melts[END_REF] are 7.1 and 6.7 wt%, respectively for the φ s = 0.38 and 0.52 suspensions. Average surfacic crystal fractions of 0.38 + 0.01 and 0.52 + 0.01 were obtained on respectively 12 and 11 scanning electron microscopy (SEM) images. Plagioclases exhibit a tabular morphology (shape ratio R~ 4, Table 1) and are chemically homogeneous in the two suspensions.

The rheological measurements were performed with a Paterson apparatus [START_REF] Paterson | Rock deformation tests to large shear strain in torsion[END_REF]. The length / diameter of the sample deformed in compression (~1) is relatively small compared to ratios used in compression tests (≥ 2; [START_REF] Hawkes | Uniaxial testing in rockmechanics laboratories[END_REF]. However, the low applied finite strain ensures a homogeneous no-slip deformation of the sample. The deformation experiments were performed at a confining pressure of 300 MPa, temperatures of 900 and 800°C for φ s = 0.38 and 0.52 respectively (corresponding to the temperatures and pressures of synthesis of each suspension), and strain rates of 1 x 10 -5 s - 1 to 1 x 10 -3 s -1 . Suspensions with φ s = 0.52 were deformed in torsion and with φ s = 0.38 in compression because the viscosity of the latter suspension was too low. To compare the apparent viscosities obtained in torsion and compression, compressive strain rates and stresses were converted to shear strain rates and stresses by using Eq. 39 and 40 from [START_REF] Paterson | Rock deformation tests to large shear strain in torsion[END_REF]. Three torsion experiments were performed (PP032, PP038, PP043), each at a different strain rate; however, for the no-slip compression experiment (PP070), a stepping strain rate procedure was applied (increase of the strain rate in a stepwise manner, once flow at constant stress is achieved).

The resulting microstructures were observed optically and with SEM on thin sections cut along the diameter, parallel to the length of the sample ([YZ] plane) in compression and normal to the radius of the core of the sample ([XZ] plane) in torsion. The long axis orientation α of the fabric was measured using the intercept method with Intercept2003 software [START_REF] Launeau | Fabric analysis using the intercept method[END_REF]. The starting suspensions have a nearly isotropic fabric, with no preferred orientation as revealed by the random distribution of the long axis orientations on the rose diagram (Fig. 1). The 2D shape fabric obtained by the intercept method has been compared with the 3D crystallographic preferred orientation (CPO) in one torsion experiment (PP043), measured using electron back-scattered diffraction (EBSD) technique [START_REF] Heidelbach | Texture analysis of a recrystallized quartzite using electron diffraction in the scanning electron microscope[END_REF].

RHEOLOGICAL MESUREMENTS AND MICROSTRUCTURAL OBSERVATIONS

The chemical composition of the plagioclase does not change appreciably during the experiments (Table 1). The normative glass compositions are also constant and H 2 O concentrations are identical within error to those in the starting suspensions. Therefore, neither crystallization nor dissolution takes place during the deformation experiments.

In the compression experiment, for each applied strain rate, a linear increase of the stress with increasing strain characterizes the first step of deformation and corresponds to the relaxation time of the sample (Fig. 2A). The higher the strain rate, the higher the stress. The peak of the stress is reached at ε ≈ 0.5% for all applied strain rates, and it is followed by flow at nearly constant stress. Two steps performed at the same high strain rates (ε& = 1 x 10 -3 s -1 ) showed similar rheological behaviour which confirms the reproducibility of the experiments.

The rheological response in the compression experiment is associated with the development of a pervasive fabric oriented at α = -3° (Fig. 2B). No discrete structures, such as shear zones or bands, are observed In the torsion experiments, a peak of the differential stress is reached at shear strains of γ ≈ 0.1 (Fig. 2C). Iit is followed by a nearly steady state to γ ≈ 0.4 and then by a decrease of the shear stress with strain, observed up to γ = 1.4 (Fig. 2C), characteristic of strain weakening. This behaviour differs from those observed for quasi isometric particles where a stabilisation of stress with respect to strain occurs after the relaxation time [START_REF] Caricchi | Non-Newtonian rheology of crystal-bearing magmas and implications for magma ascent dynamics[END_REF][START_REF] Champallier | Experimental investigation of magma rheology at 300MPa: From pure hydrous melt to 75[END_REF]. This rheological behaviour is coupled with the development of a strong pervasive shape fabric of crystals at α = 43° with respect to the shear direction (Fig. 2D, zone 1). This fabric is crosscut by 40-µm-thick shear zones, spaced at 600 µm, oriented at α = -20° and flanked by symmetrical shear gradients indicating a dextral sense of shear, synthetic to the bulk deformation (Fig. 2D, zone 2). According to [START_REF] Fernandez | Theoretical and experimental study of fabrics developed by different shaped markers in two-dimensional simple shear[END_REF], a fabric orientation of 43° is theoretically reached for γ ~ 0.1-0.15, which corresponds to the peak stress of our rheological curves. As this orientation is still observed at the final strain, we can assume that the development of the penetrative fabric is stopped at γ ~ 0.15 and the development of discrete parallel shear bands begins. The fabric inside the shear zone is oriented close to the bulk shear direction (α = -3°) and not to the side of the shear band, regardless of the finite strain. This indicates that the fabric inside the shear band likely corresponds to a transtensional shear zone geometry in which the extensional flow apophyse is always parallel to the bulk simple shear. Plagioclase crystals inside and outside the shear zone have the same size, aspect ratio and chemical composition, and are identical to those in the starting suspension (Table 1).

The CPO of the plagioclases in the pervasive fabric (Fig. 2E) shows a maximum concentration of {010} poles at ~ 45° counterclockwise with respect to the shear plane, and girdles of {001} and {100} at ~ 60° and 50° clockwise. This indicates a dominantly planar fabric, with the trace of the {010} plane in the [XZ] plane oriented near 45° clockwise, similar to α (43°). In the shear zone, plagioclases {010} poles are oriented perpendicular to the bulk shear plane, as observed from the shape fabric. Both the {001} and {100} pole figures show a girdle parallel to the shear plane, with a maximum close to the bulk shear direction, defining a plano-linear crystallographic fabric.

DISCUSSION

The rheology of our experimental magmatic suspensions can be fitted by an Ostwald De Waele power-law equation [START_REF] Ostwald | Concerning the function rate of the viscosity of dispersion systems: IV[END_REF] of the form, in compression:

n σ ε ∝ & (1)
and in torsion:

n τ γ ∝ & (2)
where n is the stress exponent. Values of n close to 3 were measured for the two crystal fractions investigated. This suggests a non-Newtonian, shear-thinning rheological behaviour, already observed for quasi isometric and anisometric particles [START_REF] Lavallée | Non-Newtonian law for highly crystalline dome lavas[END_REF][START_REF] Caricchi | Non-Newtonian rheology of crystal-bearing magmas and implications for magma ascent dynamics[END_REF][START_REF] Caricchi | Rheological properties of magma from the 1538 eruption of Monte Nuovo (Phlegrean Fields[END_REF][START_REF] Champallier | Experimental investigation of magma rheology at 300MPa: From pure hydrous melt to 75[END_REF][START_REF] Cordonnier | Rheological properties of dome lavas: Case study of Unzen volcano[END_REF]. The apparent viscosities of each suspension η app are calculated using the maximum stress σ and τ, and the strain rate ε& and γ& , using either :

ε σ η & / = app (3) in compression and γ τ η & / = app (4) in torsion.
We introduce the relative viscosity η r defined as the ratio between the apparent viscosity app η (directly measured) and the viscosity of the interstitial melt 0 η . Therefore, η r measures the influence of crystallinity parameters (e.g., crystal fraction and shape) on the rheology. The relative viscosities obtained in this study at different strain rates and crystal fractions are shown in Figure 3. At constant strain rate (1 x 10 -4 s -1 ), the relative viscosity increases by about 1 log unit (10 5.65 to 10 6.48 ) when the crystal fraction increases from φ s = 0.38 to 0.52. Furthermore, the strain weakening induces a decrease of the viscosity of about 0.25 log unit for φ s = 0.52.

Our relative viscosities can be compared with experimental results on suspensions of quasi isometric particles at strain rates similar to those of this study [START_REF] Caricchi | Non-Newtonian rheology of crystal-bearing magmas and implications for magma ascent dynamics[END_REF][START_REF] Champallier | Experimental investigation of magma rheology at 300MPa: From pure hydrous melt to 75[END_REF]Fig. 3). Both types of suspensions are characterized by a stress exponent close to 3: increasing the strain rate by 1 log unit leads to a decrease of the relative viscosity by about 0.7 + 0.1 log unit (Fig. 3). However, at given crystal fractions and strain rates, viscosities in this study are about 5 log units higher than those obtained on suspensions of quasi isometric crystals (alumina, quartz). The Einstein-Roscoe equation, which fits quite well the rheological data for the quasi isometric suspensions, fails to reproduce our data by several orders of magnitude (Fig. 3). We emphasize that the size of crystals differs by about one order of magnitude between this and the experimental deformation studies above [START_REF] Caricchi | Non-Newtonian rheology of crystal-bearing magmas and implications for magma ascent dynamics[END_REF][START_REF] Champallier | Experimental investigation of magma rheology at 300MPa: From pure hydrous melt to 75[END_REF]. However, the size of crystals, although not systematically explored, is generally considered as a second-order parameter on suspension rheology [START_REF] Mueller | The rheology of suspensions of solid particles[END_REF]. Therefore, the critical parameter controlling the relative viscosities in the two groups of studies is the shape of crystals. Elongated particles are able to generate a crystal network and thus increase the viscosity of the suspension at low crystal fractions [START_REF] Saar | Numerical models of the onset of yield strength in crystal-melt suspensions[END_REF].

Our experimental microstructures are comparable to certain natural magmatic textures. At low crystal fractions (φ s < 0.4), a pervasive fabric develops by crystal rotation and translation [START_REF] Fernandez | Theoretical and experimental study of fabrics developed by different shaped markers in two-dimensional simple shear[END_REF] and this leads to the stabilisation of the stress with increasing strain, as observed in our experiments (Fig. 2A). This corresponds to alignments and tiling of feldspar crystals in igneous rocks [START_REF] Vernon | A practical guide to rock microstructure[END_REF]. Such crystal fabrics are commonly used as kinematic indicators of early magmatic flow [START_REF] Philpotts | Magmatic flow-direction indicators in a giant diabase feeder dike[END_REF].

With increasing the crystal fraction (φ s ~ 0.5), strain partitioning occurs, with the development of two associated fabrics [START_REF] Vigneresse | Strain partitioning during partial melting and crystallizing felsic magmas[END_REF]. These discrete structures, called "trachytic textures" in feldspar rich lava [START_REF] Smith | Structural analysis of flow-related textures in lavas[END_REF], are geometrically identified to S/C'-like orientations and used in the field as shear sense indicators [START_REF] Nicolas | Kinematics in magmatic rocks with special reference to gabbros[END_REF]. They have been recently re-interpreted as asymmetric textural domains due to quasiviscous flow deformation on the flanks and bases of trachyte lava domes (Závada et al., 2009) and along conduit walls.

Our results can be directly applied to the evaluation of viscosity changes associated with the crystallization of microlites during ascent of andesitic magmas in volcanic conduits.

This process yields groundmasses with as much as 50 vol% microlites, essentially plagioclase (e.g., [START_REF] Burgisser | Pre-explosive conduit conditions of the 1997 Vulcanian explosions at Soufrière Hills Volcano, Montserrat: II. Overpressure and depth distributions[END_REF]. The change in relative viscosity associated with microlite crystallization can be estimated to be at least 5 orders of magnitude (Fig. 3).

Assuming simple Poiseuille flow and a system free of gas bubbles, this implies a reduction of the magma ascent rate in the same proportions. This would promote the formation of a magma plug and the overpressurization of the underlying magma column, eventually leading to an explosive eruption.

CONCLUSION

Our high temperature high pressure experimental deformation tests provide the first documentation of the influence of high fractions of anisometric particles (plagioclase) on the rheology of synthetic magmatic suspensions. Upon increasing the crystal fraction, the magmatic suspension evolves from a nearly steady-state flow to a strain weakening behaviour, this change being correlated with a microstructural evolution from a pervasive to a strain-partitioning fabric. These results stress (1) the interplay between rheology and the development of microstructures and (2) the importance of the shape of crystals on the mechanical behaviour of the suspensions. Magmatic suspensions of plagioclase have viscosities approximately five orders of magnitude higher than suspensions of equivalent crystallinities made of isometric particles such as quartz.

Závada, P,. Schulmann, K., Lexa, O., Hrouda, F., Haloda, J., and Týcova, P., 2009, The mechanism of flow and fabric development in mechanically anisotropic trachyte lava:

Journal of Structural Geology, v. 31, p. 1295-1307.

FIGURE CAPTIONS

Figure 1 .

 1 Figure 1. SEM microphotograph of a plagioclase bearing suspension before deformation with φ s = 0.52. Plagioclase in light grey, silicic glass in dark grey and gas bubbles in dark. The

Figure 2 .

 2 Figure 2. Rheology and microstructures of plagioclase-bearing suspensions deformed in

Figure 3 .

 3 Figure 3. Relative viscosities of magmatic suspensions plotted as a function of the crystal fraction above φ s = 0.3. Data for anisometric crystals (plagioclase) from this study

ACKNOWLEDGEMENTS O. Rouer and A. Genty helped with the SEM and F. Heidelbach helped with the electron backscattered diffraction (EBSD) analyses. L. Peilleron provided useful technical assistance. This study was funded by the French programmes ACI Fabriques magmatiques (ACI JC3013) and ANR-EXPLANT (ANR-05-CATT-003). The European Union Research Infrastructure Transnational Access (#131) programme supported the EBSD analyses at Bayreuth.


Eric Serre

Bertrand Viaud

Towards a

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J Douin

O Henry

F Dabosi

Ph Sciau

TEM characterization of the fine scale microstructure of a Roman ferrous nail

Keywords: Iron, Microstructure, Transmission Electron Microscopy, Archaeometallurgy, Roman period,

This paper describes the microstructure of a Roman ferrous nail through its observation by transmission electron microscopy. The morphologies of pearlitic colonies and ferritic grains are detailed and the relationship between pearlitic colonies and ferrite in Roman nails is explicitly demonstrated for the first time. Observations also confirm the presence of dislocations in ferritic grains and attest to the existence of very small carbide precipitates that have not been pointed out previously in standard archaeometric studies.

Introduction

Iron is one of the materials that has most contributed to the economical, sociological and scientific evolution of humankind. Pure iron is not a hard material and iron metallurgy only became critical when techniques to improve its mechanical properties were discovered. Steel gets most of its attractive mechanical properties from adequate alloying of iron with carbon, but also from the controlled conditions of the elaboration process, leading to large changes in the fine microstructure, thus the mechanical properties. For a long time, iron was not produced by the same procedure as modern steel, which undergoes complex but well documented processes. Little is known on the sub-micronic microstructure of ancient irons. In Western Europe, from the Iron Age to the production process using a form of cast iron, also called pig iron, i.e. a time span of about 2500 years, there has been little change in the basic operation of iron manufacturing by direct reduction of iron ores [1][2][3][START_REF] Mangin | Le fer[END_REF][START_REF] Wayman | [END_REF].

Nails are par excellence common artefacts of the Roman period. Roman nails have been previously studied by many authors (see for example [6][7][8][9]). Studies have shown that, at that time, iron production and processing used to produce nails had become pre-industrial and were already playing a significant part of the economic and military development. The socalled bloomery process, also referred to as "direct reduction", was the original method of producing iron. It operates on a small scale with charcoal and at relatively low temperatures, firstly in bowl and then in schaft furnaces, giving a spongious loop of more or less malleable iron due to its carbon content polluted by slags and residual charcoal pieces. The reduction reactions are complex in the wide range of temperatures and atmospheres encountered in the shaft furnace, with for the former a maximum value of 1250-1300°C, much lower than the melting point of iron (1532°C). So, the reduction occurs in solid state with separation of a pasty metallic sponge. The slags, issued from the gangue minerals (silicates, alumina and lime), with a melting point around 1200°C, are liquid and usually eliminated by pouring them out of the furnace through a hole in the bottom part of its wall. Then, the iron loop is refined in a refinery forge: the blacksmith has to remove the slag and charcoal inclusions from the metallic mass through repetitive hammering and reheating in order to make the carbon steel more homogeneous and malleable. Then the metal is usually hot-worked by forging wrought bars that can be reshaped into various implements. It must be just mentioned here that, later in Europe, around the 14 th and 15 th centuries A.D., much higher temperatures of 1700°C or more were reached with the blast furnace. The associated process, called indirect reduction, involves the initial production of liquid cast iron, very brittle after solidification, with 3 to 5%wt of carbon that must be properly decarburized for producing steel or iron.

To our knowledge, all the investigations on such archeological irons were done on a scale where very fine details of the microstructure are not observable, at most using scanning electron microscopy (SEM). For example, the observation of dislocations or nanometric carbide precipitates, which play a major role in mechanical properties, requires a better resolution. Moreover, because there is a strong heterogeneity in the carbon content of iron crystallites only, it is the purpose of this paper to explore and discuss the new information on ancient nails obtained by transmission electron microscopy (TEM), at a sub-micronic scale, as it has already been carried out successfully in another field of cultural heritage materials [10].

Experimental

We have worked on iron nails (Fig. 1) extracted during excavations at Puy d'Issolud (France) where the Uxellodunum battle took place in 52 BC, during the Gallic Wars [START_REF] Gilliver | Caesar's Gallic Wars 58-50 BC[END_REF][START_REF] Renoux | [END_REF]. These nails were subject to standard archaeometry studies [START_REF] Renoux | [END_REF]. Figure 2 shows an optical micrograph after chemical etching to reveal the variation of the grains structure from the surface to the inside of the nails. Near the surface, we observe the typical Widmanstätten structure. On the middle of the micrograph, pearlitic colonies are observed. The inner part of the nail seems essentially constituted by ferritic grains separated by carbides that have precipitated at the grains boundaries. TEM samples were extracted from the inner parts of the nails.

In order to reduce magnetization of the material in the TEM, small squared samples of about 1 mm and 220 µm thick were sliced from the nails, referred here as PM98, and subsequently mechanically ground to a thickness of 25 ± 5 µm. The samples were then electropolished with a Struers A2 electrolyte at -13°C in a Tenupol III apparatus. The thin foils were then examined using a JEOL 200 CX electron microscope operating at 200 kV equipped with a double tilt goniometer stage. From the optical micrographs (fig. 2) it was assumed that the microstructure was essentially formed by ferritic grains, TEM reveals that it is formed by a mixture of grains of ferrite (α) and colonies of pearlite, the latter being a lamellar mixture of ferrite and of the cementite iron carbide Fe 3 C. The microstructure is not very homogeneous as the relative density of pearlitic and ferritic areas varies in the different observed thin foils obtained from different parts of the nails.

Results and Discussion

General microstructure

When slowly cooled down, alloys containing less than 0.8 wt% carbon, formed hypoeutectoid ferrite from austenite, in the range 910°C-723°C with enrichment of the residual austenite in carbon, until at 723°C the remaining austenite, now containing 0.8% wt carbon transforms to pearlite (for a comprehensive review, see [START_REF] Bhadeshia | Steels[END_REF]). The three components -ferrite, cementite and pearlite -are then the principal constituents of the microstructure of plain carbon steels, provided they have been subjected to relatively slow cooling rates to avoid the formation of metastable phases, i.e. martensite or bainite. No metastable phase has been evidenced during our observations, which attests that the thermodynamical equilibrium has been reached during the production process.

Pearlitic colonies

Some areas of the microstructure are dominated by pearlitic colonies (Figure 4). The orientation of the cementite lamellae are usually related to the orientation of an adjacent austenite grain [START_REF] Bhadeshia | Steels[END_REF], thus present various different orientations depending of the neighboring grain.

When observed with a higher magnification, the cementite laths appear very well organized within a colony, with a constant orientation and separation distance (Figures 4 and5). This is particularly clear when the laths are seen end-on, an observation that allows to determine the average thickness of the laths: l = 40 ± 3 nm. The orientation relationship (OR) between the Fe 3 C lamellae and the ferritic matrix can be determined directly by TEM observations. Two different relationships are usually ascribed to exist in pearlite, the Pitsch/Petch [START_REF] Pitsch | [END_REF]15] and the Bagaryatski [16] relationships, and often side by side in the same steel. Our observations show that only the Pitsch/Petch relationship is present in the PM98 nail. This is exemplified in Figure 6, which presents a colony of cementite laths embedded in a ferritic matrix, with the (001) plane of the cementite laths parallel to the ! 5 21 ( ) of the ferrite matrix. According to the Pitsch/Petch relationship [14, 15]: (100) c at 2.6° from

! 13 1 [ ] α (010) c at 2.6° from ! 113 [ ] α (001) c // ! 5 21

( )α

When observed along the !

012

[ ] axis of the ferritic matrix (Fig. 6b), the selected area diffraction pattern of this area presents characteristic features of the Pitsch/Petch relationship: the diffraction pattern from the cementite phase (in black) is characteristic of its thin foil in a TEM allows diffracted beams to be observed even when not perfectly perpendicular to the incident beam provided the foil is thin enough [START_REF] Hirsch | Electron Microscopy of thin crystals[END_REF]. ( ) plane of ferrite is parallel to the (001) c plane of the cementite phase.

Pitsch/Petch relationship happens when pearlitic nodules nucleate on clean austenite boundaries, while Bagaryatski relationship was found to hold for pearlite nodules nucleated on hyper-eutectoid cementite. It is then predicted that Pitsch/Petch-type colonies predominate as the true eutectoid composition is approached, whereas Bagaryatski-type colonies should prevail at higher carbon levels. According to [START_REF] Bhadeshia | Steels[END_REF], the interlamellar spacing is inversely related to the temperature of the pearlite formation: the measured mean spacing of 0.17 µm corresponds to a processing temperature of 900 K for an alloy with 0.8% C, i.e. for eutectoid pearlite. This gives an estimate of the temperature at which the transformation occurred during forging.

On the mechanical point of view, the presence of pearlite strengthens the steel. This is directly related to the confinement of the deformation in the ferrite lamellae: since cementite is hardly deformable, cementite laths form obstacles to the movement of the dislocations, constricting their motion in ferrite lamellae. Strength of pearlite is then expected to increase as the interlamellar spacing is decreased, and the interlamellar spacing is inversely proportional to the degree of undercooling. The interlamellar spacing is assumed to be constant for a given alloy and a given transformation temperature. This is valid for plain carbon steel where the average composition of the pearlite is identical to that of the austenite from which it grows. In the case presented in Fig. 6, the average distance between the habit planes of the laths is d = 0.17 ± 0.02 µm, which contributes to an increase to the strength of about µ b/d = 2.10 -3 µ (where b is the modulus of the Burgers vectors of activated dislocations, and µ the shear modulus of ferrite).

Ferritic grains

Due to the absence of cementite laths, dislocations may more freely be created and move in pure ferritic grains. Indeed, ferritic grains are found with a significant density of dislocations usually interacting to each others (Fig. 7a) attesting that the material has been deformed during and/or after the processing. The dislocation density ρ ranges from 10 12 to 10 13 m -2 , significantly smaller than the usual dislocations density of 10 15 -10 16 m -2 of modern steels, which undergo strong deformation (e.g. lamination) during the material processing. This dislocations density contributes through the forest hardening mechanism to an increase of the strength of the ferritic grains of αµbρ 1/2 , i.e. approximately 4.10 -4 µ (with α = 0.5). Notice also that this moderate dislocation density in the ferritic grains, as well as the absence of strong stress concentrations, pile-ups, walls of dislocations and/or cracks within the observed microstructure, indicate that the ferritic grains will continue to deform upon stress and then still appear as the ductile part of this material.

When observed with a higher magnification (Fig. 7b), the frequent occurrence of dislocation loops also attests of the presence of obstacles to the dislocation motion. As the material was slowly cooled down and not quenched, these loops couldn't have been produced by vacancies condensation. There is an old debate of whether or not the formation of small loops is related to cross-slip in such materials. Cross-slip activity can be observed during deformation of iron. However, in pure iron there is no loop formation (see for example the very recent work by Caillard, [START_REF] Caillard | [END_REF]19]), while loops formation is favored by the presence of impurities (see for example in Fe-3%Si [20]). Cross-slip activity is strongly helped by the presence of obstacles, thus the observation of dislocations loops attest to the presence of small areas (that we suppose to be small carbides here) that act as obstacles to the dislocation motion. Such dislocations behavior is frequent when they interact with an obstacle during their motion: by-passing of an obstacle, often helped by cross-slip, is activated, resulting in the activation of the Orowan process and the formation of a dislocation loop around the obstacle.

The presence of such obstacles to the dislocation motion is also responsible for material strengthening, by limiting the dislocations ability to move under the applied external stress.

The increase in hardening can be evaluated as µb/L, where L ≈ 0.2 µm is the mean distance between precipitates, i.e. about 10 -3 µ for the ferritic grains.

Estimation of the different contributions to the strength of the material

Assuming a shear modulus µ of 200 GPa, the hardening due to pearlitic colonies can be estimated to about 400 MPa. Taking the yield strength of pure iron as about 150 MPa, the yield stress of a pearlitic colony should then be close to 550 MPa. In ferritic grains, dislocations and obstacles contribute to an increase in strength of about 80 MPa and 200 MPa, respectively, that is a yield stress of about 430 MPa for ferritic grains.

These values can be correlated with those of Vickers microhardness values H V obtained on these ancient nails [START_REF] Dabosi | presented at the International Archaeometry Symposium[END_REF]. For ferritic structures, mean values of H V number, under a low load of 50g., stay between 100 and 130 for grain sizes of 70 and 30 micrometers, respectively, while in ferritic-pearlitic (hypoeutectoïd) area, H V reaches usually 200. On a rough approximation, the relation between H v and the yield stress is σ y = H v /3. Thus, after conversion in MPa, Vickers microhardness values give an approximated yield stress of 375 MPa for ferritic grains and 650 MPa for pearlitic grains, which compare well, and at least have the right order of magnitude, with the values deduced from TEM observations. A posteriori, these numbers confirm that the pearlitic grains are more difficult to deformed, thus that ferritic grains form the most ductile part of the material.

Conclusion

Transmission Electron Microscopy observations allowed for the gathering of information at a sub-micronic scale on ancient iron materials. These observations could not have been obtained using standard techniques typically used in archaeometallurgy, scanning electron microscopy for example.

While the production process of the nails used direct reduction, and thus occurred in solid state, the observed microstructure is close to what is obtained through the modern carbonsteels process, involving intermediary steps of liquid cast iron. This results from a forging process at a temperature high enough to allow the formation of a mixture of pearlitic colonies and ferritic grains at thermodynamical equilibrium [START_REF] Bhadeshia | Steels[END_REF].

The microstructure of the pearlitic colonies has been described precisely and for the first time in this category of ancient materials: the orientation relationship between cementite laths and ferrite is of the Pitch/Petch type. The average distance between cementite laths ranges from 0.15 to 0.2 µm. The direct observation of a sizeable density of dislocations in ferritic grains confirms that the material has been mechanically work-hardened, presumably by hammering during nail forging. Strengthening nanometer-scale precipitates in ferritic grains have also been clearly pointed out. Our observations show that, amongst the different strengthening mechanisms, both the presence of regularly-spaced cementite lamellae in pearlitic colonies and the existence of nanometer-scaled obstacles in ferritic grains contribute significantly to the strength of the material, furthermore their contributions to the yield stress have been numerically estimated in agreement with Vickers hardness values.

Finally the microstructure details obtained thanks to the TEM technique give significant information concerning the formation process of these artifacts, which cannot be obtained from standard archaeometrical investigations. Also, the sample preparation does not present particular difficulties and does not require removing a large amount of material compared to the sample preparations made for abrasive standard techniques used in archaeometallurgy. It is therefore clear that, in addition to the standard archaeometallurgical techniques, TEM can be a powerful means to study the internal microstructure of these artefacts, giving information on the temperature and kinetic formation of individual grains as well as the mechanical properties from the dislocation network.

Fig. 1 .

 1 Fig. 1. Nails from the Uxellodunum site from which the PM98 steel samples have been selected (Photo courtesy of G. Renoux and F. Dabosi)

Fig. 2 .

 2 Fig. 2. Optical micrograph of the PM98 nail. On the left, inner part of the nails with ferritic grains and carbides precipitated at the grains boundaries; in the center, pearlitic colonies; near the surface, Widmanstätten structure. The dark layer on the right part of the micrograph is the epoxyde used for sample preparation.

Figure 3

 3 Figure 3 shows general views of the microstructure of the inner part of PM98 Roman nail by TEM.

Fig. 3 .

 3 Fig. 3. TEM observation of the microstructure of the PM98 nail showing intricate pearlitic colonies (P) and ferritic areas (F) (limits are underlined).

Fig. 4 .

 4 Fig. 4. Area of the PM98 nail dominated by pearlitic colonies with various orientations.

Fig. 5 .

 5 Fig. 5. One pearlitic colony showing the arrangement of the parallel cementite laths over a large range scale. Notice the very regular spacing between cementite lamellae.

! 1 10 (

 10 ) plane, with the 004 spot from cementite perfectly aligned with the ! 5 21 spot of ferrite, and the 220 spot of cementite only slightly misaligned from the 121 spot of ferrite. Notice that the spots of the systematic 220 row of the cementite are visible here due to the fact that diffraction of a

Fig. 6 .

 6 Fig. 6. (a) Micrograph of a pearlitic colony and (b) the corresponding selected area diffraction pattern (inverted contrast): in white the

Fig. 7 .

 7 Fig. 7. a) Micrograph of a ferritic grain attesting of the large density of dislocations impeached by small obstacles and large carbide precipitates. b) Enlargement of the area indicated in (a) attesting of the frequent occurrence of dislocation loops (arrows) surrounding small obstacles, according to the Orowan by-pass process.

Acknowledgements

The authors would like to thank G. Renoux who had the kindness to commit us the artefact specimens, and J. Crestou for his valuable help in samples preparation.

This study was partly supported by the PPF "Le fer dans les sociétés anciennes des régions du sud", under contract MEN-MSTP 2007-2010.


Dr Catherine François

Veronique Descamps

Etienne Brochot

Isabelle Bernard

Valerie Canva

Philippe Mathurin

Sandrine Castelain

Gilles Duverlie

Catherine ; Francois
email: catherine.francois@u-picardie.fr

Véronique Descamps

Valérie

Relationship between the hepatitis C

Keywords: HCV, IFN, INTERFERON, HEPATITIS C, PHARMACOKINETIC interferon, hepatitis C, HCV, IFN, pharmacokinetics 42

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

Fifteen treatment-naive patients with chronic hepatitis C infection (genotypes 1, 2, 3 and 4) 29 underwent PEG-IFNα-2b/ribavirin combination therapy. Blood samples were collected before 30 the first injection (T 0 ) and then at different time points until the next injection a week later. 31

The PEG-IFN concentration and the HCV load were assayed. 32

The serum interferon concentration peaked two days after the first injection (mean value for 33 the study population; T max =40.9 hours; C max =490 pg/ml) and a trough in viral load was seen at 34 day three. The PEG-IFNα-2b concentration decreased from day 2 to day 7, enabling a viral 35 rebound in all patients. The change in viral load between day 0 and day 3 differed 36 significantly according to whether the patients were responders at week 12 37 (∆logD0/D3=2.729±1.419 Log 10 IU/mL) or not (∆logD0/D3=1.102±0.472 Log 10 IU/mL). 38

Our results emphasize the potential clinical importance of achieving viral decay immediately 39 after initiation of interferon-ribavirin combination therapy. 40

Introduction 44

The hepatitis C virus (HCV; family: Flaviviridae; genus: Hepacivirus) is a major causative 45 agent of chronic hepatitis -a condition that can progress to cirrhosis and hepatocellular Given that novel drugs (such as protease and polymerase inhibitors) will not become available 67 in the very near future, efforts are being made to optimize current therapies and thus increase 68 the response rate. The first parameter to have been modified was the treatment duration. 69 Whereas a shorter course of treatment will be sufficient for some patients, longer treatment is 70 advisable for others [START_REF] Francois | Optimizing the 252 treatment of chronic viral hepatitis C[END_REF][START_REF] Mangia | 283 Individualized treatment duration for hepatitis C genotype 1 patients: A randomized 284 controlled trial[END_REF]. These new therapeutic 71 strategies are based on evaluating the early-stage viral kinetics in general and the HCV load 72 one month after treatment initiation in particular. Indeed, the rapid virologic response (RVR, 73 defined as an undetectable viral load after 4 weeks of treatment) is highly predictive of the 74 sustained response rate [START_REF] Martinot-Peignoux | Virological response at 4 weeks to predict outcome of hepatitis C treatment with 292 pegylated interferon and ribavirin[END_REF]. Hence, analysis of the change in 75 viral load over the first month may be helpful for modifying the treatment as soon as possible, 76 as a function of the observed antiviral efficacy. 77

In order to establish whether or not the early treatment response is a useful parameter, we 78 studied the pharmacokinetics of PEG-IFNα-2b and HCV levels during the first week of 79 treatment in 15 patients and evaluated the correlation between these two parameters. 80

Patients and Methods 82

Samples: 83

Serum samples were obtained from 15 patients chronically infected with HCV (genotype 1: 84 n=10; genotype 2: n=1; genotype 3: n=1; genotype 4: n=3). All patients were interferon-naïve 85 and eligible for combination therapy (Table 1). HCV RNA had been detectable for at least the 86 previous year. The treatment was once weekly 1.5 µg/kg PEG-IFNα2b, combined with daily, 87 oral ribavirin given according to bodyweight recommendations and regardless of the genotype 88 (1000 mg/day for patients under 75 kg and 1200 mg/day for those over 75 kg). Enrollment 89 began in 2003. 90 Blood samples were collected before (T 0 ) the first injection and then 1, 2, 3, 5 and 8 hours, 2, 91 3, 4, 5, 8, 15 and 29 days and 8 and 12 weeks afterwards. Samples were processed within four 92 hours of collection and stored at -70°C. The treatment outcome was defined as the response at 93 week 12, which is considered by the French consensus group as a good time point for 94 predicting non-response to combination treatment [START_REF] Dhumeaux | Treatment of hepatitis C. The 2002 French 243 consensus[END_REF]. The study design 95 and protocol were approved by the local independent ethics committee. Prior to enrolment in 96 the study, all patients gave their written informed consent to participation. 97 98 Cell line: 99 MDBK-t2 cells (containing the human interferon-inducible MxA promoter linked to a 100 chloramphenicol acetyl transferase (CAT) reporter gene) were a kind gift from Dr Martin D. 101

Fray [START_REF] Fray | Validation of an Mx/CAT reporter gene assay for 254 the quantification of bovine type-I interferon[END_REF] For each assay, MDBK-t2 cells were plated at a density of 1 x 10 6 per well in six-well plates. 108 After 18H of incubation at 37°C in 5% CO 2 , the culture medium was discarded and replaced 109 with 1 mL of DMEM with 10% fetal bovine serum and 100 µL of either a sample or known 110 amounts of PEG-IFNα-2b (Schering-Plough, NJ). The cells were cultured for an additional 111 24H at 37°C in 5% CO 2 . The standard curve was constructed by serial 1:2 dilutions (from 625 112 pg/mL down to 19.5 pg/mL) of PEG-IFNα-2b. Chloramphenicol acetyl transferase expression 113 was determined as previously described [START_REF] Francois | 248 Quantification of different human alpha interferon subtypes and pegylated interferon 249 activities by measuring MxA promoter activation[END_REF]. The intra-assay and inter-114 assay coefficients of variation were 8.6% and 14.4%, respectively. The assay's limit of 115 detection (LOD) was 19.5 pg/mL [START_REF] Francois | 248 Quantification of different human alpha interferon subtypes and pegylated interferon 249 activities by measuring MxA promoter activation[END_REF]. 116 117

Determination of HCV RNA levels: 118

To prepare total RNA from sera, we used the QIAamp RNA kit (Qiagen,Courtaboeuf,119 France) according to the manufacturer's instructions. Next, HCV RNA was quantitated 120 specifically by a real-time RT-PCR assay, as described previously [START_REF] Castelain | TaqMan amplification system with an internal positive 237 control for HCV RNA quantitation[END_REF] The concentration of peginterferon-α α α α2b 129

The serum concentration of PEG-IFNα-2b was measured as described in the Materials and 130 Methods section. PEG-IFNα-2b activity was generally detected between two and three hours 131 after the injection. One patient was excluded because the PEG-IFN concentration was below 132 the LOD throughout the first week (Figure 1B). In the remaining 14 patients, the peak serum 133 concentration (C max ) of PEG-IFNα-2b (490 ± 219.8 pg/ml) was observed 2 days after the 134 injection (T max : 40.9 ± 21.9 hours) (Figure 1A). This peak was followed by a linear decline in 135 the serum PEG-IFNα-2b concentration over the next few days. On day 8 (i.e. just before the 136 scheduled injection of the next dose), the PEG-IFNα-2b concentration had fallen to below 137 100 pg/ml for ten of the 14 patients and was even below the LOD in seven cases (50%). In 138 three cases, the second injection had been performed before collection of the day 8 blood 139 sample (Figure 1B). The same decline was observed over the next three weeks for the five 140 patients for whom a blood sample was collected before the subsequent injections (data not 141 shown). Only two patients had a detectable IFN concentration in the serum before the 142 treatment. 143 144 HCV load. 145 HCV RNA was quantitated in a real-time RT-PCR assay, as described in the Materials and 146 Methods section. All 14 patients responded to the first dose of PEG-IFNα-2b, as evidenced 147 by a decrease in the viral load. During the first week, the lowest viral load was obtained on 148 day three for all patients (Fig. 2A). In order to characterize the interferon's antiviral activity, 149 we calculated the difference in viral load between day 0 and day 3 (expressed in log 10 units 150 (∆log)). As shown in Figure 2B, the mean ∆log d0/d3 was 1.820 ± 1.203 for genotype 1 or 4 151 comparison was made for responders vs. non-responders at week 12 (i.e. no detectable HCV 153 RNA at week 12). The mean ∆log d0/d3 values were 1.102 ± 0.472 and 2.729± 1.419 for the 154 non-responder and responder patients, respectively (p=0.0388) (Fig. 2C). The same trend was 155 observed when considering only genotype 1 patients, although the ∆log d0/d3 values did not 156 differ significantly (2.57±1.19 and 1.69±0.96 for responders and non-responders, 157 respectively; n=10; p=0.28). As shown in Table 2, all the patients with a ∆log d0/d3 > 2 were 158 responders at week 12 and 80% of these individuals showed a sustained virologic response. 159 160

The relationship between HCV load and IFN concentration during the first week of 161

treatment. 162

The treatment's antiviral activity was visualized by plotting the HCV load and the IFN 163 concentration over the first week of treatment on the same graph and for all patients (Figure 164 3A). The two parameters were inversely correlated, with a correlation coefficient of -0.794 165 (p<0.01) (Figure 3B). There was a decrease in the HCV load when the PEG-IFN 166 concentration was at its highest; conversely, the fall in IFN concentration was accompanied 167 by a rebound in the HCV load. 168 One of today's main goals in monitoring the treatment for chronic HCV infection is to predict 171 the SVR as soon as possible after initiation of the therapy. This knowledge may usefully 172 enable therapy to be discontinued in patients who will ultimately fail to benefit or, on the 173 contrary, may help patients to maintain good compliance if the treatment appears to be 174 effective. Hence, understanding the early pharmacokinetics and pharmacodynamics of IFN 175 could be useful for predicting the treatment response. 176

Here, we first looked at pharmacokinetic parameters. Our results confirm previous reports on 177 the pharmacokinetics of PEG-IFNα-2b [Zeuzem et al., 2003]. The serum PEG-IFN 178 concentration peaked at day 2 post-injection and then fell slowly over the course of the week. 179

Hence, in contrast to what had been expected [START_REF] Wang | Structural and 317 biological characterization of pegylated recombinant interferon alpha-2b and its 318 therapeutic implications[END_REF], the PEG-IFNα-2b 180 concentration did not remain stable over the week as a whole. At the end of the week (i.e. 181 before the next scheduled injection) serum IFN could not be detected in most patients. These 182 findings confirmed a number of other reports [START_REF] Bruno | Viral 228 dynamics and pharmacokinetics of peginterferon alpha-2a and peginterferon alpha-2b 229 in naive patients with chronic hepatitis c: a randomized, controlled study[END_REF][START_REF] Formann | Twice-weekly administration of 245 peginterferon-alpha-2b improves viral kinetics in patients with chronic hepatitis C 246 genotype 1[END_REF]. 183

When IFN was no longer detectable in the serum, the viral load increased until the next IFN 184 injection. This observation suggests that the antiviral effect of IFN is not optimal during the 185 first week of treatment and that the pharmacokinetic parameters could be improved. Indeed, 186 this is why twice-weekly injection of PEG-IFNα-2b has been suggested by Formann and 187 collaborators [START_REF] Formann | Twice-weekly administration of 245 peginterferon-alpha-2b improves viral kinetics in patients with chronic hepatitis C 246 genotype 1[END_REF]]. In the latter report, the blood PEG-IFNα-2b 188 concentration was maintained throughout the week and the HCV rebound was avoided. These 189 results were confirmed by Bruno and collaborators, who compared the change in HCV RNA 190 load with the pharmacokinetics of PEG-IFNα-2a and PEG-IFNα-2b [START_REF] Bruno | Viral 228 dynamics and pharmacokinetics of peginterferon alpha-2a and peginterferon alpha-2b 229 in naive patients with chronic hepatitis c: a randomized, controlled study[END_REF]. At 191 week 12, the HCV load was significantly lower in the PEG-IFNα-2a-treated group than in the 192 PEG-IFNα-2b-treated group -suggesting that the difference in HCV RNA values may be 193 PEG-IFNα-2b. However, the latter study was performed by testing the patients' serum during 196 the first week of treatment and did not necessarily reflect pharmacodynamics of interferon in 197 the liver [START_REF] Bruno | Pharmacodynamics of peginterferon alpha-2a and peginterferon 233 alpha-2b in interferon-naive patients with chronic hepatitis C: a randomized, 234 controlled study[END_REF]. 198

The significance of the fact that the trough in HCV load was seen 24 hours after the peak 199 serum IFN and that a HCV rebound occurred rapidly after the IFN concentration had 200 decreased is subject to debate. However, this finding could mean that IFN-induced host 201 proteins with a short half-life could be involved in the early antiviral response. 202 Both viral and host factors have been implicated in the response to IFN therapy. In terms of 203 the viral factors, genotype seems to be an important predictive factor. In particular, genotypes 204 1 and 4 show higher levels of resistance to IFN treatment. Conversely, genotypes 2 and 3 are 205 more sensitive to IFN therapy [START_REF] Fried | 257 Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection[END_REF][START_REF] Manns | Peginterferon alfa-2b plus ribavirin 287 compared with interferon alfa-2b plus ribavirin for initial treatment of chronic 288 hepatitis C: a randomised trial[END_REF]. The present study 206 confirmed that the early decline in viral load (∆log d0/d3) is lower for genotypes 1 and 4 than 207 for genotypes 2 and 3. However, this difference was not statistically significant -no doubt due 208 to the low number of patients in general and genotype 2 or 3 patients in particular. Several 209 lines of evidence now suggest that early viral clearance in patients infected with HCV is a 210 sign of more effective IFN-ribavirin combination therapy [START_REF] Bekkering | Estimation of 225 early hepatitis C viral clearance in patients receiving daily interferon and ribavirin 226 therapy using a mathematical model[END_REF]Galeras JA, 211 2006;[START_REF] Jessner | Early viral kinetics on treatment with pegylated interferon-alpha-2a 280 in chronic hepatitis C virus genotype 1 infection[END_REF][START_REF] Rosen | 307 Early hepatitis C viral kinetics correlate with long-term outcome in patients receiving 308 high dose induction followed by combination interferon and ribavirin therapy[END_REF]. The analysis of the ∆log d0/d3 appears to 212 further corroborate this hypothesis. Just as the rapid virologic response is predictive of the 213 sustained response rate [START_REF] Jensen | Early identification of HCV genotype 1 patients 276 responding to 24 weeks peginterferon alpha-2a (40 kd)/ribavirin therapy[END_REF][START_REF] Martinot-Peignoux | Virological response at 4 weeks to predict outcome of hepatitis C treatment with 292 pegylated interferon and ribavirin[END_REF]Yu et al., 2007] 214 [START_REF] Deltenre | A 2-240 log drop in viral load at 1 month is the best predictor of sustained response in HCV 241 patients with normal ALT: a kinetic prospective study[END_REF], the ∆log d0/d3 might also be an early-stage parameter with predictive 215 value. However, the present results must be verified with a large number of patients.

  . The cells were maintained in Glutamax I-Dulbecco's modified Eagle 102 medium (DMEM) supplemented with 10% fetal bovine serum and 10 µg/mL blasticidin 103(Invitrogen, Cergy Pontoise, France)

  4.115 ± 0.757 for genotype 2 or 3 patients (p=0.0679). The same 152

  different absorption and distribution profiles of the two drugs. No differences in 194 gene induction were observed when comparing the pharmacodynamics of PEG-IFNα-2a and 195

Figure 2

 2 Figure legends 327

Journal of Medical Virology

Acknowledgements 219 220

We thank David Fraser for English corrections. 221 222


Christine Larsen
email: c.larsen@invs.sante.fr

Vanina Bousquet

Elisabeth Delarocque-Astagneau

Corinne Pioche

Françoise Roudot-Thoraval

Jean-Claude Desenclos

Jean- Claude Desenclos

C Sylvain

D Capron

P Cales

Angers I Hubert-Fouchard

V Di

J Foucher

Clermont- A Abergel

M P Ripault

V Loustaud-Rati

P Pradat

C Trepo

J J Raabe

( Metz

D Larrey

J Gournay

E Marine-Barjoan

A Tran

Hepatitis C virus genotype 3 and the risk of severe liver disease in a large population of drug users in France

Keywords: alcohol, cirrhosis, drug users, HCV genotype 3, HIV alcohol, cirrhosis, drug users, HCV genotype 3, HIV

come

Hepatitis C virus genotype 3 and the risk of severe liver disease in a large population of drug users in France

Introduction 1

In France, the prevalence of anti-HCV in injecting drug users ranges from 55% to 60% [Jauffret-2 Roustide et al., 2009; [START_REF] Meffre | Prevalence of hepatitis B and hepatitis C virus infections in France in 12 2004: Social factors are important predictors after adjusting for known risk factors[END_REF]. In addition, injecting drug users accounted for more than 3 two thirds of the patients co-infected with HIV and HCV [START_REF] Larsen | Prevalence of hepatitis C and hepatitis B infection in the 12 HIV-infected population of France[END_REF].

4

Factors associated with progression of liver fibrosis have been well established, and are related to host 5 factors (age at contamination, male sex), comorbidities (co-infection with HIV, heavy alcohol 6 consumption), metabolic conditions (obesity, steatosis, diabetes mellitus) and duration of infection 7 [START_REF] Mallat | Environmental factors as disease accelerators 1 during chronic hepatitis C[END_REF][START_REF] Massard | 7 Natural history and predictors of disease severity in chronic hepatitis C[END_REF]. Furthermore, co-infection with HIV and a history of accounting for more than one third [START_REF] Payan | Changing of hepatitis C virus genotype patterns in France at the 6 beginning of the third millenium: The GEMHEP GenoCII Study[END_REF]. Excessive alcohol consumption is common in 14 this population, too [START_REF] Campbell | High prevalence of alcohol use among hepatitis C virus antibody 7 positive injection drug users in three US cities[END_REF]]. However, limited data are available on factors associated 15 with progression to liver fibrosis in former or current injecting drug users infected by HCV. [START_REF] Prasad | Little evidence that 17 hepatitis C virus leads to a higher risk of mortality in the absence of cirrhosis and excess 18 alcohol intake: the Swiss Hepatitis C Cohort Study[END_REF] In addition, with new antiviral treatment options and efficacy, recommendations on antiviral treatment 17 of drug users infected with HCV have been expanded in France [START_REF] Dhumeaux | Treatment of hepatitis C. The 2002 French 11 consensus[END_REF] The database was checked regularly for duplicate reporting in and between participating reference 7 centres.

8

The suspected year of HCV infection was defined by the year of the last HCV negative test performed 9 during the drug-use period or year of first drug injection. Time from the suspected year of infection to 10 the year of referral to the reference centre was used as a surrogate of the duration of HCV infection.

11

The median duration of infection was used to create a qualitative variable (≥ 18 years; < 18 years) in 12 the univariate and multivariate analysis.

13

Patients included in the analysis were drug users with HCV RNA in the serum who were naive to 14 anti-HCV treatment.

15

The outcome of severe liver disease was defined as cirrhosis (compensated or not) or hepatocellular 16 carcinoma at the first referral, based on the clinical assessment.

23

First, the general approach for multivariate analysis was used including only patients who had no 24 missing data in any of the variables (complete case analysis). However, six out of the eight variables Of the 4236 patients positive for HCV RNA, 77% were male; the median age was 39 years and the 15 median estimated duration of HCV infection was 18 years (Table I). Co-infections with HBV and 16 HIV were diagnosed in 2% and 6%, respectively. A history of excessive alcohol consumption was 17 reported for 43 %. Among the 1154 patients who reported previous heavy drinking in the past with an 18 available duration of excessive consumption, 67% had drunk heavily for more than five years. HCV 19 genotypes were mainly genotype 1 (36%) and 3 (25%), the distribution of HCV genotypes remained 20 stable over the study period. genotyping result: HCV genotypes 1 and 3 were found in 48% and 42%, respectively. Of the 164 1 patients with severe liver disease at clinical evaluation who underwent a liver biopsy, the Metavir 2 score F4 was diagnosed for 94% including three out of the four patients with hepatocellular carcinoma 3 (F3 for the remaining patient).

4

In univariate analysis the following variables were associated with a severe liver disease at referral 5 (Table II): inclusion after 2003, male sex, age >40 years at referral, estimated duration of infection 6 ≥18 years, history of excessive alcohol intake, co-infection with HIV and HCV genotype 3. Time 7 between the first HCV positive test and referral was not significant.

8

In both the complete-case and the multiple imputation (MI) analyses, the independent factors 9 associated with severe liver disease at clinical evaluation were male sex (adjusted OR MI (aOR MI ): 2.0; 10 95% CI, 1.4-2.8), age > 40 years at referral (aOR MI : 2.1; 95% CI, 1.6-2.9), duration of HCV infection 11 ≥ 18 years (aOR MI : 2.9; 95% CI, 2.0-4.3), history of excessive alcohol consumption (aOR MI : 2.8; 95% 12 CI, 2.1-3.7) and HCV genotype 3 (aOR MI : 1.6; 95% CI, 1.2-2.1) (Table III). In the multiple 13 imputation analysis, co-infection with HIV (aOR MI : 1.8; 95% CI, 1.2-2.8) was associated significantly 14 with severe liver disease. No significant interactions were found.

15

The univariate analysis for the factors associated with the F4 Metavir score, limited to the patients 16 who had a liver biopsy (n=1725), showed similar results although the 95% confidence intervals were 17 wider. However, co-infections with HIV and HBV were not significant statistically (p>0.25; data not The analysis of the mortality data in the Swiss hepatitis C cohort showed recently that mortality was 1 increased for individuals who reported drinking more than 40g alcohol per day. HCV genotype 3 was 2 associated also with a higher mortality although it did not remain significant in the multivariate 3 analysis [START_REF] Prasad | Little evidence that 17 hepatitis C virus leads to a higher risk of mortality in the absence of cirrhosis and excess 18 alcohol intake: the Swiss Hepatitis C Cohort Study[END_REF].

4

HIV infection is known to modify the natural history of HCV by accelerating the rate of progression 5 of fibrosis and the development of advanced fibrosis both before [START_REF] Poynard | A comparison of 15 fibrosis progression in chronic liver diseases[END_REF]] and since 6 [START_REF] Thein | Natural history of hepatitis C virus infection in 9 HIV-infected individuals and the impact of HIV in the era of highly active antiretroviral 10 therapy: a meta-analysis[END_REF] the highly active antiretroviral therapy era. Interestingly, a study has shown 7 advanced liver fibrosis to be associated with HCV genotype 3 in a population of patients co-infected 8 with HIV and HCV [START_REF] Barreiro | Predictors of liver fibrosis in HIV-infected patients with 11 chronic hepatitis C virus (HCV) infection: assessment using transient elastometry and the role 12 of HCV genotype 3[END_REF]. Surprisingly, in the study by [START_REF] Barreiro | Predictors of liver fibrosis in HIV-infected patients with 11 chronic hepatitis C virus (HCV) infection: assessment using transient elastometry and the role 12 of HCV genotype 3[END_REF], neither 9 past nor current excessive alcohol consumption remained associated significantly with advanced liver 10 fibrosis. However, the assessment of liver fibrosis in this study was based on a non-invasive method 11 (elastometry) only. 12 Among the limitations of the present study is its cross-sectional design. Indeed, it is difficult to use The present analysis was confronted with missing values as it was based on surveillance data. In order 10 to keep all cases in the analysis, a multiple imputation method was applied to the data. Multiple 11 imputation is a well established method for the analysis of data sets with missing values. As the 12 multivariate analysis will include all cases, multiple imputation provides consistency in results [Little 13 & Rubin, 2002]. Thus, the association between HCV genotype 3 and severe liver disease found in the 14 complete case analysis was reinforced by the multiple imputation analysis. Also, HIV infection was 15 associated significantly with severe liver disease after multiple imputation analysis. In addition, the 16 sensitivity analysis performed on HCV genotype 3, co-infection with HIV and alcohol consumption 17 allowed to be confident of the robustness of the estimations [START_REF] Carpenter | Sensitivity analysis after multiple imputation 9 under missing at random: a weighting approach[END_REF]

8 excessive

 8 alcohol consumption are linked to death at an earlier age in patients infected chronically 9 with HCV as shown in a recent study on mortality related to chronic hepatitis C in France [Marcellin 10 et al., 2008]. Viral induced steatosis linked to HCV genotype 3 has also emerged as a cofactor for 11 progression of liver fibrosis in patients infected with HCV [Rubbia-Brandt et al., 2004]. HCV 12 genotype 3 is also the most frequent genotype found among drug users infected with HCV in France,

13

 13

since the 18 2002 11 Data collection 12 A

 181112 consensus conference on treatment of HCV infection.19 In France, a surveillance system was set up to monitor changes over time of the epidemiological and 20 clinical characteristics of patients infected with HCV at the first referral to hepatology reference 21 centres; nearly 3900 cases of chronic hepatitis C, of whom 35% acknowledged drug use, have been 22 recorded each year since 2001 by this system. The aim of the present study was to assess risk factors Details of the methods have been described previously [Delarocque-Astagneau et al., 2005]. Briefly, 1 this surveillance system based on voluntary participation of 26 of the 30 existing hepatology reference 2 centres was set up in 2000 to monitor changes over time of the epidemiological and clinical 3 characteristics of patients infected with HCV at the time of their first referral. Hepatology reference 4 centres are located in University hospitals throughout France. To be included, a case was defined as a 5 patient with anti-HCV antibodies who was referred (first contact as an in-or out-patient) to any 6 participating reference centre. Patients can be referred by their general practitioner, by a specialist or 7 by self-referral. Patients included in the analysis were those who reported having injected or snorted 8 drugs at least once in their lifetime. To ascertain the first referral, patients with no prior histological 9 evaluation of their liver disease at referral were selected for the analysis. The surveillance system 10 obtained ethical approval from the French data protection authority (CNIL). standardized notification form was used in all centres to collect data routinely, including risk 13 factors for HCV transmission, the last HCV negative test (if available), date and the reasons for 14 anti-HCV testing, HCV RNA serum status, HCV genotype, co-infections with HIV (anti-HIV 15 serostatus) and hepatitis B virus (hepatitis B surface antigen serostatus), history of excessive alcohol 16 consumption defined as more than 210g of pure ethanol per week for women and 280g for men, and 17 the number of years with excessive consumption. The severity of liver disease was assessed either by 18 liver biopsy (Metavir scores [Bedossa and Poynard, 1996]) and/or by clinical evaluation including 19 clinical examination, biochemical tests and imaging (mainly abdominal ultrasound). The clinical 20 evaluation was graded as follows: repeated normal ALT values defined as ALT levels below the 21 upper limit of normal at three consecutive tests within six months, chronic hepatitis C, cirrhosis 22 (compensated or not) and hepatocellular carcinoma. Cirrhosis at clinical evaluation was defined by 23 the fibrosis Metavir score F4 when a liver biopsy was carried out or by the association of clinical 24 signs, laboratory findings and ultrasound images used by all participating centres [Schuppan and 25 Afdhal, 2008]. The diagnostic criteria used for hepatocellular carcinoma were those established 26 during the Barcelona-2000 EASL Conference [Bruix et al., 2001]). modified in 2004 to include data on non-invasive assessment of liver fibrosis and on 1 antiviral therapy.

2 HCV

 2 genotyping was carried out by the referral laboratories of the participating centres. Most of these 3 laboratories were involved in the French ANRS panel quality control study in 2000 [Lefrere et al., 4 2004] and the same standardized genotyping assays (Inno LiPA assay) or other methods (sequencing, 5 primer-specific PCR) during the study period were used.

6

 6

17

 17

Statistical analysis 18 Factors

 18 associated with severe liver disease 19 Univariate analysis was carried out to assess the characteristics associated with severe liver disease at 20 clinical evaluation. Variables with p values <0.2 in univariate analysis were entered into the 21 multivariate analysis. STATA® 9.2 statistical package was used for the analysis (Stata Corporation, 22 College Station, Texas, USA).

  25 included in the multivariate model comprised 10% to 27% of missing values resulting in a loss of 26 power and precision, complication in data handling and analysis and potentially biased odds ratios 27 (OR) due to differences between the observed and unobserved data [Little and Rubin, 2002]. values were estimated from the observed values using multiple imputation 1 (imputation by chained equations, ice procedure STATA® 9.2) [Royston, 2007]. Several complete 2 data sets (m=30) were created. Each data set was analysed separately, and the results were combined 3 to obtain the odds ratios [Little & Rubin, 2002]. The sensitivity of the odds ratios to departures from 4 the assumed missing mechanism was tested for three variables (alcohol consumption, co-infection 5 with HIV and HCV genotype 3) using a weighting approach developed by Carpenter et al. drug users who were referred for HCV infection between 2001 and 2007 and who were 10 naive to anti-HCV treatment, 4236 were HCV RNA positive and were included in the analysis. The 11 remaining 768 had either a negative (n = 455) or an unspecified (n = 313) result for HCV RNA.

21 A

 21 liver biopsy was carried out in 53.6% of the drug users included in 2001-2003 and in 27.1% of 22 those included in 2004-2007. In the latter period, 29.2% had an evaluation of fibrosis by non-invasive 23 methods only.

24A

  clinical evaluation of liver disease was available for 4065 (96%) patients with HCV RNA, and 25 severe liver disease was diagnosed in 8% of the patients. Cirrhosis was diagnosed in 301 patients and 26 hepatocellular carcinoma in 10 patients. Of these 311 patients, 236 (76%) had an available HCV 27

  site study documents factors associated with severe liver disease in a large group of 22 patients infected with HCV who reported drug use. The findings confirm the role of excessive alcohol 23 consumption and co-infection with HIV and suggest that HCV genotype 3 is associated with an 24 increased risk of severe liver disease independently of age, sex, duration of infection and excessive 25 alcohol consumption. To our knowledge this is the first study to address the risk factors of severe liver 26 disease in a population of drug users infected with HCV at first referral.

10 [

 10 Clinical studies have demonstrated a significant association between HCV genotype 3 infection and 1 the presence of steatosis[START_REF] Cross | The impact of hepatic steatosis 18 on the natural history of chronic hepatitis C infection[END_REF][START_REF] Hui | 1 Genotype-specific mechanisms for hepatic steatosis in chronic hepatitis C infection[END_REF][START_REF] Rubbia-Brandt | Steatosis affects chronic hepatitis C progression in a genotype 4 specific way[END_REF]. In patients 2 infected with HCV genotype 3, fat accumulation in the liver is correlated with the level of HCV 3 replication [Adinolfi et al., 2001] and disappears following successful antiviral therapy [Castera et al., 4 2004]. Although there have been several studies providing evidence for the influence of steatosis on 5 the progression of fibrosis regardless of the HCV genotype [Cross et al., 2009; Fartoux et al., 2005; 6 Hu et al., 2004] or in patients infected with HCV genotype 3 [Castera et al., 2003; Castera et al., 2004; 7 Rubbia-Brandt et al., 2004], the issue remains controversial. Indeed, other studies failed to show any 8 association between steatosis and liver fibrosis [Asselah et al., 2003; Perumalswami et al., 2006; 9 Ryder et al., 2004], including one study on a large cohort of patients infected with HCV Perumalswami et al., 2006]. However, the comparability of the studies is questionable, as there were 11 differences in the study populations and the risk factors studied. Furthermore, another study showed 12 that steatosis had no association with fibrosis in patients infected with HCV genotype 3 [Bugianesi et 13 al., 2006] suggesting other causes for the progression of fibrosis such as insulin resistance or viral 14 induced liver inflammation. 15 Numerous studies have shown that a past or present history of excessive alcohol consumption was 16 associated with more rapid progression to fibrosis [Tolstrup et al., 2009; Westin et al., 2002]. As 17 expected in this particular population of patients who reported drug use, cirrhosis at the time of first 18 referral was associated strongly with excessive alcohol consumption in the past. The definition of 19excessive alcohol consumption used in this study corresponds to more than 30g/day for women and 20 40g/day for men compared with 50g/day for both sexes in many other studies. Of those who 21 acknowledged excessive alcohol consumption in the past, more than half reported that the period 22 lasted at least five years, allowing the development of fibrosis. In addition, more than one third of the 23 patients who reported excessive alcohol consumption in the past, acknowledged current excessive 24 consumption. This is consistent with the results reported by Campbell et al.[START_REF] Campbell | High prevalence of alcohol use among hepatitis C virus antibody 7 positive injection drug users in three US cities[END_REF], 25 who found that in a population of drug users infected with HCV, knowledge about the detrimental 26 effects of alcohol consumption related to HCV infection did not appear to influence alcohol use.

13 cross- 5 2002

 135 sectional data to estimate longitudinal parameters. However, although collected at the same time 14 as the outcome of interest (severe liver disease), most of the variables assessed in this study reflect 15 exposures that occurred prior to the outcome. Therefore, this study could be considered as a 16 retrospective cohort, which is less subject to bias than a purely cross-sectional study[Rothman and 17 Greenland, 1998]. Others factors shown to be associated with progression of fibrosis were not 18 collected, such as daily cannabis smoking[START_REF] Hezode | Daily cannabis smoking as a risk factor for 16 progression of fibrosis in chronic hepatitis C[END_REF] and insulin resistance related to the 19 patient characteristics such as obesity and type 2 diabetes mellitus, and to the virus [Cua et al., 2008; 20 Moucari et al., 2008; Petta et al., 2008]. However, Moucari et al. [2008] provided evidence that 21 insulin resistance was associated with HCV genotypes 1 and 4 in contrast to patients with HCV 22 genotypes 2 and 3. This lower rate of insulin resistance in patients with HCV genotype 3 was shown 23 also in another study [Hui et al., 2003]. The validity of the method used in the present study for 24 assessing the severity of liver disease could be called into question since it was not based on 25 histology. However, the classification used for clinical evaluation of the liver disease is likely to be 26 homogeneous within the participating reference centres. Furthermore, as the indication for liver 27 biopsy may be related to certain risk factors or the intention to begin a course of antiviral therapy, the 28 clinical stage for assessment of severe liver disease appeared more suited to the purpose of 1 the study objectives. Also, the proportion of patients managed in a reference centre who had a liver 2 biopsy decreased dramatically from 45% in 2001 to 18% in 2006 [Delarocque-Astagneau et al., 3 2009]. Indeed, since 2002, physicians in France have been treating patients infected with HCV 4 genotypes 2 and 3 or with HIV co-infection without performing a liver biopsy, in accordance with the French recommendations on HCV treatment [Dhumeaux et al., 2003]. The univariate analysis of 6 the factors associated with the F4 Metavir score limited to patients who had a liver biopsy showed 7 similar results, but the 95% confidence intervals were wider and co-infection with HIV was not 8 significant statistically.

9

 9

18 A

 18 recent analysis of the data from the Swiss hepatitis C cohort found HCV genotype 3 as a factor 19 associated with progression of liver fibrosis, which reinforced the validity of the results [Bochud et 20 al., 2009].21These results suggest that in patients infected chronically with HCV who reported drug use, early 22 therapeutic intervention should be recommended for those patients who are infected with HCV 23 genotype 3 and who acknowledge excessive alcohol consumption in the past, since progression to 24 cirrhosis may be more rapid. Therefore, integrated treatment of HCV and alcohol addiction is 25 essential. Thus, collaboration between healthcare providers who specialize in the management of 26 addictions and those who specialize in the management of HCV infection should be promoted.

4a

  Time from suspected year of infection to year of referral to the reference centre. Suspected year of HCV 5 infection is defined as year of the last HCV negative test performed during the drug-use period or year of 6 first drug injection 7 b >210g/week for women and >280g/week for men (g = gram of ethanol)

  [data not shown].

Table I :

 I Main characteristics of HCV RNA positive drug users at first referral to hepatology reference available for 3938 (93.0%) HCV RNA+ drug users, for 1023 (95.0%) infected with genotype 3, for 1906 (96.0%)

	1 1 2 2 3 4 5	Note. Data are no. (%) of patients or median values (interquartile range), unless otherwise indicated. HCV, hepatitis centres in France, 2001-2007. C virus; IQR, interquartile range; SD, standard deviation; ALT, alanine aminotransferase; ULN, upper limit of All patients HCV genotype 3 other HCV genotypes normal; HIV, human immunodeficiency virus; HBsAg, hepatitis B surface antigen; a available for 3626 (85.6%) HCV RNA+ drug users, for 932 (86.5%) infected with genotype 3, for 1673 (84.2%) Characteristic (n = 4236) (n = 1077) (n = 1986) infected with other genotype;
	6 7 8 9 10	Sex ratio (female/male) Age, years Duration of HCV infection a , years, ALT level b , xULN infected with other genotype; c >210 g/week for women and >280 g/week for men (g= gram of ethanol) 1 / 3 39 (10) 18 (12) 1.5 (1.5) 2.0 (1.9) 1 / 3 39 (11) 18 (12) d including decompensated cirrhosis	1 / 3 40 (10) 19 (12) 1.5 (1.2)
		History of excessive alcohol intake c
		Missing		467 (11.0)	119 (11.1)	212 (10.7)
		No		1952 (46.1)	499 (46.3)	908 (45.7)
		Yes		1817 (42.9)	459 (42.6)	866 (43.6)
		HIV serostatus
		Missing Negative Positive HBsAg serostatus Missing Negative Positive HCV genotype	702 (16.6) 3284 (77.5) 250 (5.9) 685 (16.2) 3466 (81.8) 85 (2.0) e r F o F o r P P e	162 (15.0) 868 (80.6) 47 (4.4) 150 (13.9) 906 (84.1) 21 (2.0)	289 (14.6) 1 557 (78.4) 140 (7.0) 285 (14.3) 1 660 (83.6) 41 (2.1)
		Missing 1 non a, b 1a		1173 (27.7) 407 (9.6) 694 (16.4) e r e r
		1b 2 3 non a 3a 4 5 Clinical stage at 1 st referral Missing Normal ALT values	439 (10.3) R R 117 (2.8) 317 (7.5) e e 760 (17.9) 325 (7.7) 4 (0.1) v i e v i e 171 (4.1) 31 (2.9) 713 (16.8) 116 (10.8) w w	58 (2.9) 356 (17.9)
		Chronic hepatitis		3000 (70.8)	815 (75.6)	1419 (71.4)
		Cirrhosis d		301 (7.1)	97 (9.0)	132 (6.7)
		Hepatocellular carcinoma	10 (0.2)	3 (0.3)	4 (0.2)
		Acute hepatitis		41 (1.0)	15 (1.4)	17 (0.9)
		Liver biopsy at 1 st referral	1765 (41.7)	405 (37.6)	992 (49.9)
		Fibrosis Metavir [Bedossa and Poynard, 1996] score at liver biopsy	1725	396	976
		F0		138 (8.0)	25 (6.3)	81 (8.3)
		F1		735 (42.6)	136 (34.3)	455 (46.6)
		F2		480 (27.8)	112 (28.3)	264 (27.1)
		F3		195 (11.3)	62 (15.7)	94 (9.6)
		F4		177 (10.3)	61 (15.4)	82 (8.4)
					21

b

Table II :

 II Factors associated with severe liver disease at clinical evaluation in HCV RNA positive 1 drug users at first referral to hepatology reference centres in France, 2001-2007 2

					Univariate Analysis
		Factors	Patients	% SLD	OR (95% CI)	p
			(n = 4065)
		Period of inclusion
		2001-2003	2211	6.8	1
		2004-2007	1854	8.7	1.3 (1.0-1.6)	0.02
		Sex
		Female	929	4.0	1
		Male	3136	8.7	2.3 (1.6-3.3)	<0.001
		Age
		≤ 40 years	2302	3.7	1
		> 40 years	1763	12.8	3.8 (2.9-4.9)	<0.001
		Time between 1 st HCV+ test and referral
		< 1 year	1683	6.8	1
		≥ 1 year	1987	7.7	1.1 (0.9-1.5)	0.3
		Missing	395	10.6
		Duration of HCV infection at referral a
		< 18 years	1636	3.0	1
		≥ 18 years	1843	12.5	4.7 (3.4-6.5)	<0.001
		Missing	586	7.7
		History of excessive alcohol intake b
		No	1881	4.1	1
		Yes	1740	12.5	3.3 (2.5-4.3)	<0.001
		Missing	444	3.6
		HBsAg
		Negative	3366	7.8	1
		Positive	83	13.2	1.8 (0.9-3.4)	0.07
		Missing	616	6.0
		HIV
		Negative	3180	7.7	1
		Positive	240	12.9	1.8 (1.2-2.7)	0.004
		Missing	645	5.6
		HCV genotype 3
		No	1928	7.0	1
		Yes	1046	9.6	1.4 (1.1-1.8)	0.02
		Missing	1091	6.9
	3	Note. SLD, severe liver disease (cirrhosis, hepatocellular carcinoma); aOR, odds ratio; CI, confidence
		interval; HCV, hepatitis C virus; HBsAg, hepatitis B surface antigen; HIV, human immunodeficiency virus;

Journal of Medical Virology

Acknowledgments

28

This work was done within the surveillance activities of the Institut de veille 4 sanitaire that is funded by the French Ministry of Health.

5 V. Bousquet is funded by a PhD grant from the Agence Nationale de Recherches sur le Sida et les 6 hépatites virales (ANRS).