C Ferrier-Pages

A Peirano

M Abbate

S Cocito

A Negri

C Rottier

Pascal Riera

R Rodolfo-Metalpa

S Reynaud

C Ferrier-Page

Summer autotrophy and winter heterotrophy in the

HAL is

Cladocora caespitosa is a Mediterranean scleractinian coral (Faviidae family), comparable to its tropical congeners because it is colonial, lives in symbiosis with dinoflagellates commonly called zooxanthellae, and can form large banks or extensive bioherms (Kruz ˇic ´and Poz ˇar-Domac 2003; Kruz ˇic ´and Benkovic ´2008; Peirano et al. 2004). This coral has, however, a wider habitat range, from well-lit shallow-water environments to relatively dark conditions (no more than 20 mmol photons m 22 s 21 ) such as those occurring at 40-m depth (Peirano 2007) or in the turbid waters of La Spezia (Italy, Eastern Ligurian Sea), which receives loads of sediment from the Magra plume (Peirano et al. 1999). Laboratory experiments have shown that its association with zooxanthellae can supply most of its carbon requirements, except under low light conditions, under which the coral has to rely more on the heterotrophic feeding [START_REF] Schiller | Ecology of the symbiotic coral Cladocora caespitosa (Favidae, Scleractinia) in the Bay of Piran (Adriatic Sea). II. Energy budget[END_REF][START_REF] Hoogenboom | Co-variation between autotrophy and heterotrophy in the Mediterranean coral Cladocora caespitosa[END_REF]. C. caespitosa, and corals in general, are indeed able to increase their feeding effort when prey are available and photosynthesis is suppressed or lowered [START_REF] Anthony | Shifting roles of heterotrophy and autotrophy in coral energy budgets at variable turbidity[END_REF][START_REF] Rottier | Experimental assessment of the feeding effort of three scleractinian coral species during a thermal stress: Effect on the rates of photosynthesis[END_REF][START_REF] Hoogenboom | Co-variation between autotrophy and heterotrophy in the Mediterranean coral Cladocora caespitosa[END_REF]. They can prey on a wide range of particles, from bacteria and dissolved organic matter to macrozooplankton (reviewed in Houlbre `que and Ferrier-Page `s 2008). This heterotrophic mode of nutrient acquisition is particularly important in temperate symbiotic anthozoans, because they often live in habitats with lower light levels, colder temperatures, and higher nutrient concentrations compared to tropical areas (Muller-Parker and [START_REF] Muller-Parker | Temperate and tropical algal-sea-anemone symbiosis[END_REF]. Studies performed on the temperate sea anemone Anthopleura elegantissima [START_REF] Engebretson | Translocation of photosynthetic carbon from two algal symbionts to the sea anemone Anthopleura elegantissima[END_REF][START_REF] Bergschneider | Nutritional role of two algal symbionts in the temperate sea anemone Anthopleura elegantissima Brandt[END_REF] or on the temperate coral Astrangia [START_REF] Szmant-Froelich | The effects of feeding frequency and symbiosis with zooxanthellae on the biochemical composition of Astrangia danae Milne Edwards[END_REF]Pilson 1980, 1984;[START_REF] Dimond | Temporal variation in the symbiosis and growth of the temperate scleractinian coral Astrangia poculata[END_REF], both from the Pacific, indeed concluded that during unfavorable conditions, the host derives most of its nutrition via heterotrophy. The question is therefore to know if heterotrophy plays the same important role for theM e d i t e r r a n e a nc o r a lC. caespitosa. Although this issue has been assessed in two laboratory experiments with artificial food, supplied in high concentrations (Rodolfo-Metalpa et al. 2008b;[START_REF] Hoogenboom | Co-variation between autotrophy and heterotrophy in the Mediterranean coral Cladocora caespitosa[END_REF], the natural heterotrophic carbon and nitrogen acquisition of this species is still unknown.

The aim of this study was therefore to investigate the extent of auto-and heterotrophy in colonies of C. caespitosa living under natural conditions and to determine the potential food sources for this coral. This knowledge will bring a better understanding of the functioning of temperate symbioses, and of their response to stresses (Rodolfo-Metalpa et al. 2006, 2008c), which usually induce a decrease in symbiont density, as well as in photosynthetic capacities and autotrophic carbon acquisition (Rodolfo-Metalpa et al. 2006).

In coastal environments, however, there are generally a large number of food sources for benthic consumers, and the diversity in the origin of the organic matter makes it difficult to determine which are the main food sources. Stable carbon and nitrogen isotope ratio (d 13 C and d 15 N) analysis has proven to be a useful tool in delineating carbon flows and trophic relationships in a large variety of marine ecosystems [START_REF] Fry | d 13 C measurements as indicators of carbon flow in marine and freshwater ecosystems[END_REF][START_REF] Peterson | Stable isotopes in ecosystem studies[END_REF], and serves as a long-term index of carbon (and nitrogen) assimilation (Muscatine et al. 1989;[START_REF] Bergschneider | Nutritional role of two algal symbionts in the temperate sea anemone Anthopleura elegantissima Brandt[END_REF]. While carbon isotope composition is, in many cases, characteristic of each primary source of organic matter [START_REF] Farquhar | Carbon isotope discrimination and photosynthesis[END_REF]) and the 13 C: 12 C ratio of consumers is similar to those of their diet [START_REF] Deniro | Influence of diet on the distribution of carbon isotopes in animals[END_REF], the enrichment factor in d 15 N between two consecutive trophic levels is higher, from 2.3% to 3.4% [START_REF] Minagawa | Stepwise enrichment of 15 N along food chains: Further evidence and the relation between d 15 N and animal age[END_REF][START_REF] Mccutchan | Variation in trophic shift for stable isotope ratios of carbon, nitrogen and sulfur[END_REF]. Isotopic analyses of anthozoan tissue have already been used, either in tropical species (Muscatine et al. 1989;[START_REF] Muscatine | Resource partitioning by reef corals as determined from stable isotope composition. II. d 15 N of zooxanthellae and animal tissue versus depth[END_REF][START_REF] Reynaud | Effect of feeding on the carbon and oxygen isotopic composition in the tissues and skeleton of the zooxanthellate coral Stylophora pistillata[END_REF] or in a temperate sea anemone [START_REF] Bergschneider | Nutritional role of two algal symbionts in the temperate sea anemone Anthopleura elegantissima Brandt[END_REF], to estimate the importance of heterotrophy in their diet. This technique has, however, never been applied to temperate corals, except in cold-water corals [START_REF] Carlier | Trophic relationships in a deep Mediterranean cold-water coral bank (Santa Maria di Leuca, Ionian Sea) Mar[END_REF], which are fundamentally different from shallowwater symbiotic ones.

To study the nutritional mode of C. caespitosa, and to assess the productivity of the zooxanthellae and their contribution to the animal host, several colonies were sampled, in both winter and summer, from three localities in the Ligurian Sea (northwest Mediterranean Sea) where this species is particularly abundant (Peirano et al. 1999). The isotopic signature of the coral tissue was compared to the signature of the zooxanthellae, its autotrophic source of nutrition, and to the signature of the particles in seawater (from bacteria to macrozooplankton) and sediment, which are its potential heterotrophic food sources.

Methods

Study site-Corals were collected at ca. 8-10-m depth at three locations (Fig. 1) in the Ligurian Sea (northwest Mediterranean Sea). The first sampling site, named Lerici, was located in front of the town of Lerici (44u04.339N, 09u54.419E), and the second site, named Punta Bianca (44u02.169N, 09u58.769E), was in front of the Magra River, 8 km away from Lerici. These two sites have silt-sandy bottoms, because they are under the influence of the Magra plume, which carries high loads of particles and sediment, especially during wintertime. At these locations, colonies live in a very turbid environment and are often covered by a thin layer of sediment. Underwater photosynthetic active irradiance (PAR) at 9-m depth in both locations varied from a mean of 50 mmol photons m 22 s 21 in winter (mean seawater temperature of 13uC) to 206-230 mmol m 22 s 21 in summer (mean temperature of 23uC). The third site, called Bonassola (44u10.889N, 09u34.729E), was at ca. 50 km from Lerici. Here, corals are at the same depth, and experience the same temperatures, but they are located in clear waters and on rocky substrate. Underwater PAR therefore varies between a mean of 114-145 mmol m 22 s 21 in winter (Peirano et al. 1999) and a mean value of 400 mmol m 22 s 21 in summer. The other characteristics of these Ligurian waters are fully described in Peirano et al. (2005).

Sampling and treatments-Corals: Coral samples were collected during 2-3 d in winter (January) and summer (August) 2009. Five colonies (with ca. 30 polyps, or a total surface of 30-40 cm 2 ) were randomly selected at each site and each season. They were enclosed underwater in a plastic bag containing the surrounding seawater and brought back to the laboratory, where they were cleaned of epiphytes and sediment, divided into two samples of ca. 20 and 10 polyps respectively, and frozen until subsequent analysis.

No more than a week after sampling, the 20 polyp samples from each mother colony were thawed and individually placed in 100-mL beakers (precombusted at 480uC for at least 4 h in a ThermolyneH 62700 oven) containing 20 mL of filtered seawater. For each sample, tissue was completely removed from the skeleton with an air pick and homogenized with a Potter tissue grinder (also precombusted at 480uC for at least 4 h). Half of the homogenate was directly flash frozen and freeze-dried. It will be referred as to the symbiotic association in the following sections and represents the mean signal of both the host tissue and zooxanthellae. The other half of the homogenate was divided into a host fraction and a zooxanthellae fraction. For the host fraction, the homogenate was centrifuged (Biofuge 17RS Heraeus) at 3000 g for 10 min at 4uC to pellet most of the zooxanthellae. The supernatant was recentrifuged twice to eliminate the remaining zooxanthellae (as checked under a microscope), then transferred in 50-mL Pyrex tubes, flash frozen with liquid nitrogen, and freeze-dried using a Heto (model CT 60) drier. For the zooxanthellae fraction, the zooxanthellae pellet was washed several times with filtered seawater (following Muscatine et al. 1989), until almost no contamination by host cells was visible under the binocular, and then also flash frozen and freeze-dried. Isotopic analyses were performed on these three sets of freeze-dried samples, with each colony treated individually.

Samples from each colony containing ca. 10 polyps were used for protein (five polyps per colony) chlorophyll (Chl) and zooxanthellae (five polyps per colony) determination. For Chl and zooxanthellae measurements, coral tissue was detached from the skeleton using a Water PickH in 12 mL of 0.45-mm-filtered seawater. The slurry was homogenized using a Potter tissue grinder and a 2-mL subsample was taken for zooxanthellae density determination using the HistolabH 5.2.3 image analysis software (Rodolfo-Metalpa et al. 2006). The remaining 10-mL subsamples were centrifuged at 5000 g for 10 min at 4uC and the pellet containing the zooxanthellae was resuspended in 10 mL of pure acetone. Pigments were extracted at 4uC during 24 h. The extract was centrifuged at 10,000 g for 15 min, and Chl a and c 2 were determined according to [START_REF] Jeffrey | New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton[END_REF]. Data were normalized to the polyp surface area determined as in Rodolfo-Metalpa et al. (2006). For protein content measurement, tissue was extracted in 1 mol L 21 NaOH at 90uC for 30 min and proteins were determined using the bicinchoninic acid assay kit (Interchim). The standard curve was established with bovine serum albumin and the absorbance was measured with a multiscan bichromatic spectrophotometer (LabsystemH). Data were normalized to the polyp surface area.

Particulate organic matter: To characterize the isotopic signature of the particulate organic matter (POM) suspended in seawater, five 10-liter bottles of seawater were sampled near the corals, at each site and each season, using a NiskinH bottle. Back to the laboratory, seawater was size fractioned using an inverse filtration device to avoid cell breakage. The following size fractions were achieved: particles above 105 mm (macroplankton), between 105 and 50 mm (microplankton), between 50 and 10 mm (nanoplankton), and between 10 and 1 mm (small nanoplankton and picoplankton). Each fraction was then filtered onto precombusted (450uC for 5 h) 25-mm Whatman GF/F filters under low vacuum. To remove carbonates, filters were quickly acidified (1 mol L 21 HCl) and rinsed with distilled water. They were then freeze-dried and kept frozen until isotopic analyses. Only a few macrozooplankton cells were collected on the filters, because of their low abundance in seawater. Therefore, to get the isotopic signal of the zooplankton for the winter and summer seasons, concentrated samples were also collected in a 5-min haul using a plankton net (type WP2). A fraction of this zooplankton was counted, whereas another fraction was collected on filters and treated as described above.

Sedimentary organic matter: Muddy sediment samples were taken at each station and each season near the corals by scraping the upper 1-cm surface of sediment (Riera et al. 1996). For the measurements of stable isotopic ratios of the sedimentary organic matter (SOM), the sand was sieved to a grain size of , 50 mm to separate sand grains from most of the sedimentary POM. A subsample of approximately 5 g was freeze-dried, and ground using a mortar and pestle. Then, 200 mg was acidified with 1 mol L 21 HCl to remove inorganic carbon. In order to prevent any loss of dissolved organics, these samples were not rinsed, but the acidification was performed on a hot plate to quickly evaporate the acid, and samples were dried overnight at 50uC under a fume extractor (Riera 1998). Once dried, the sediment was mixed with Milli-Q water, freeze-dried, ground again to a fine powder, and kept frozen (280uC) until analysis.

Stable isotope analysis-Samples were analyzed for d 15 N and d 13 C using an automated nitrogen-carbon analyzer (ANCA) coupled to a Geo-20 : 20 isotope ratio mass spectrometer (SerConH). International reference materials (IAEA-600 and IAEA-CH6, International Atomic Energy Agency) were used for scale calibration of results to Vienna PeeDee Belemnite (VPDB) and air, and two different analytical quality control samples were also analyzed with each batch for quality control purposes. Precision as determined by repeat analysis of the reference materials and quality controls was better than 6 0.20% and 6 0.15% for measured d 15 N and d 13 C values, respectively. Data are expressed in the standard d unit notation where

dX~½(R sample : R reference ){1|10 3 ð1Þ
where R 5 13 C: 12 C for carbon and 15 N: 14 N for nitrogen, and reported relative to VPDB for carbon and to air N 2 for nitrogen.

Statistics and models-The isotopic signatures of the different samples (coral tissue, zooxanthellae, symbiotic association, POM, SOM, and plankton) taken at the three locations (Lerici, Punta Bianca, and Bonassola) and in two seasons (winter and summer) were compared using a threeway analysis of variance (ANOVA) and StatViewH software. In order to give an estimation of the isotopic contribution of each determined food source to the isotopic signal of the host tissue, a computer program (IsoSource) developed by [START_REF] Phillips | Source partitioning using stable isotopes: Coping with too many sources[END_REF] and freely available at http://www.epa.gov/wed/pages/models.htm was used.

Results

Colonies of C. caespitosa from the three locations contained equivalent concentrations of zooxanthellae, Chl, and protein per skeletal surface area during the winter season (Fig. 2; one-way ANOVA p 5 0.6, 0.3, and 0.2 for the protein, zooxanthellae, and Chl, respectively). In summer, colonies from Bonassola showed significant lower zooxanthellae density and Chl concentration per skeletal surface area than in Lerici and Punta Bianca (one-way ANOVA, p 5 0.016 and 0.049 for the Chl and zooxanthellae, respectively). However, the amount of Chl per zooxanthella remained stable and equal to 3-4 pg cell 21 (data not shown). Proteins were not significantly different (one-way ANOVA, p 5 0.9). Also, for Lerici and Punta Bianca, there was no significant difference between winter and summer for protein, zooxanthellae, and Chl concentrations (Fig. 2; ANOVA, p . 0.05 for all parameters), suggesting a seasonal stability. Conversely, for Bonassola, Chl and zooxanthellae concentrations were significantly lower in summer (Fig. 2; ANOVA, p 5 0. 004 and p 5 0.02, respectively) than in winter.

The carbon isotopic signature (d 13 C) of the different samples is represented in Fig. 3 1). Therefore, for the following results and discussion, a mean value of seawater POM was calculated for each location and will be considered. Also, the d 13 C signature of the entire symbiotic association (zooxanthellae + coral host) was equal to 222.57 6 1.60%, 221.24 6 0.32%, and 222.22 6 0.80% in winter and 215.8 6 0.42%, 216.31 6 0.21%, 215.71 6 1.36% in summer for corals sampled in Punta Bianca, Lerici, and Bonassola, respectively.

d 13 Cv a l u e sw e r en o td i f f e r e n tb e t w e e nl o c a t i o n s (Table 2). They highlighted a marked seasonal difference in the signature of the symbiotic association of zooxanthellae and host tissue (Fig. 3; Table 2). Indeed, the d 13 C of the symbiotic association and zooxanthellae decreased from ca. 216% and 217% in summer to ca. 221% and 222% in winter. Also, the d 13 C of the host decreased from 217% in summer to 223%, 225% and 228% in winter, depending on the location considered. Although the host tissue was significantly more depleted than the zooxanthellae in winter (from 2% to 7% depending on the location, Tukey test, p , 0.001; Fig. 3), d 13 C values of the two fractions were identical in summer (Tukey test, p 5 0.351). Also, the comparison of the d 13 Co ft h es y m b i o t i c association with the d 13 C of the coral potential food sources (POM, SOM, and plankton) showed two different trends, according to the season. In summer, the d 13 C of the symbiotic association (ca. 215%) was highly enriched compared to the food sources (220 to 23%; Fig. 3; Tukey test, p , 0.001 between host tissue and all sources). Conversely, in winter, the d 13 C signature of the symbiotic association was more depleted (222%), reaching the same low values as for POM, SOM, and plankton (ANOVA, p 5 0.11, 0.06 and 0.25, respectively).

Concerning the d 15 N signature, all POM size fractions were pooled into one fraction, as for carbon (Table 3). Overall, the nitrogen isotopic signature was different between samples, locations, and seasons (Table 3). As for carbon, results showed a marked seasonal difference in the d 15 N of the host tissue and POM (Tukey test, p , 0.001), which were significantly enriched by 3-5% in winter. Overall, all samples presented higher d 15 N values in winter than in summer. In winter, coral tissue had higher d 15 N values compared to zooxanthellae (by 0.3-2.3%), water, and sediment (by 3-6.6%; Tukey test, p , 0.01). In summer, values were comparable (Tukey test, p . 0.05), except for SOM (Tukey test, p , 0.001).

Estimation of the isotopic contribution of each food source to the isotopic signal of the host tissue [START_REF] Phillips | Source partitioning using stable isotopes: Coping with too many sources[END_REF] has given the following information: in summer, the zooxanthellae isotopic contribution ranged from 81% to 100%. The external food sources (POM, SOM, plankton) accounted for only between 0% and 18% of the isotopic host signature in all sampling locations. Conversely, in winter, zooxanthellae contributed only 0-24% to the isotopic signal of the host, while the external food sources equally contributed between 10% and 90%. No external food source was predominant in terms of contribution, except in Punta Bianca, where POM had a main contribution of 55-75%.

Discussion

In order to better understand the functioning of temperate symbioses in terms of acquisition of energy, this study has assessed, under natural winter and summer conditions, the contribution of algal symbionts and external food to the carbon and nitrogen budgets of the temperate coral C. caespitosa. Results obtained have shown that heterotrophy increased in importance in winter, and supplied, during this season, the major part of the carbon and nitrogen needs of this coral. Conversely, autotrophy seemed to play a major role in summer.

The biomass data indicated that population densities of zooxanthellae, as well as Chl contents of the colonies sampled in Lerici and Punta Bianca, were similar between summer and winter, in spite of large seasonal differences in temperature and irradiance. Such stability of the symbiosis has previously been observed in the same coral species (Rodolfo-Metalpa et al. 2008c) and in other temperate corals and sea anemones [START_REF] Farrant | Nutrition of the temperate Australian soft coral Capnella gaboensis. The role of zooxanthellae and feeding[END_REF] 2007). The fact that corals sampled in Bonassola, only 50 km away from Lerici, presented significantly lower zooxanthellae density in summer compared to the other locations suggests that algae are rather expulsed by the host when irradiance levels are high [START_REF] Fitt | Seasonal patterns of tissue biomass and densities of symbiotic dinoflagellates in reef corals and relation to coral bleaching[END_REF]Rodolfo-Metalpa et al. 2008c). Bonassola is indeed the location that receives the highest light level in summer, compared to the other two more-shaded locations (Peirano et al. 2005). The lack of seasonal variation in the Chl content per zooxanthella was also observed in Mediterranean corals (Rodolfo-Metalpa et al. 2008c) and several other temperate anthozoans [START_REF] Farrant | Nutrition of the temperate Australian soft coral Capnella gaboensis. The role of zooxanthellae and feeding[END_REF]Verde and McCloskey 2002), and contrasts with tropical corals, which often exhibit a decrease in zooxanthellae density and pigment concentrations per zooxanthella [START_REF] Fitt | Seasonal patterns of tissue biomass and densities of symbiotic dinoflagellates in reef corals and relation to coral bleaching[END_REF]. Isotopes of carbon and nitrogen have proven to be useful in assessing the importance of heterotrophy and autotrophy in corals. Concerning nitrogen isotopes, a pure heterotrophic predator is generally enriched by 2.5-4.5% compared to its prey, because of the excretion of light nitrogen ( 14 N) as waste products [START_REF] Minagawa | Stepwise enrichment of 15 N along food chains: Further evidence and the relation between d 15 N and animal age[END_REF][START_REF] Gollety | Complexity of the food web structure of Ascophyllum nodosum zone evidenced by a d 13 Ca n dd 15 Ns t u d[END_REF]. In symbiotic corals, nitrogen ingested by the host is not excreted, but rather recycled by the zooxanthellae, inducing a 1-3% enrichment of the host compared to the zooxanthellae, not to the food [START_REF] Reynaud | Effect of feeding on the carbon and oxygen isotopic composition in the tissues and skeleton of the zooxanthellate coral Stylophora pistillata[END_REF]. Such enrichment was observed in this study in winter, confirming the previous observations of an important recycling of ingested nutrients within the symbiosis. When the whole symbiotic association (host plus zooxanthellae) is however considered as ''the predator,'' there can indeed be observed, in winter, a 2.5-3.4% enrichment between the food (POM, SOM) and the symbiotic association, showing that corals derive a large fraction of their nitrogen needs from food during this season. Conversely, in summer, the isotopic signature of the host and the zooxanthellae was comparable, and it was much lower than in winter. The isotopic signature of the symbiotic association did not follow the classical enrichment values compared to the food, suggesting that corals were relying more on dissolved sources (ammonium, nitrate) absorbed by the zooxanthellae than on the organic nitrogen derived from particulate feeding.

d 13 C values confirmed our conclusions based on d 15 N isotopes. In summer, the host and zooxanthellae presented the same d 13 C signature, suggesting that most of the host's carbon originated from its algae. Indeed, the zooxanthellae signature was significantly higher than those of the POM, SOM, and zooplankton, because of their intense photosynthetic activity of the zooxanthellae and partly because of the use of bicarbonate with a high d 13 C signature (0.8%; [START_REF] Reynaud | Effect of feeding on the carbon and oxygen isotopic composition in the tissues and skeleton of the zooxanthellate coral Stylophora pistillata[END_REF]. Inside the coral, bicarbonate undergoes several processes before reaching the photosynthetic site of the zooxanthellae, such as dehydration, which produces a large fractionation of 27% [START_REF] Deuser | Carbon isotope fractionation in the system CO 2 (gas)-CO 2 (aqueous)-HCO 2 3 (aqueous)[END_REF]. It is also mixed with some respiratory carbon, leading to a mean isotopic signature of 210% to 216% in a wide range of zooxanthellae living in tropical coral species (Muscatine et al. 1989;[START_REF] Risk | Crosscontinental shelf trends d 13 C in corals of the Great Barrier Reef: Further consideration of the reef nutrient paradox[END_REF][START_REF] Reynaud | Effect of feeding on the carbon and oxygen isotopic composition in the tissues and skeleton of the zooxanthellate coral Stylophora pistillata[END_REF]. Because the d 13 C signal of C. caespitosa was very close to the signature of tropical corals, known to mostly be autotroph for carbon, it is likely that autotrophy is also and Muller-Parker 2008). In winter, the host d 13 C signature was highly depleted compared to its summer time value or to the zooxanthellae d 13 C, reaching the low values of POM, SOM, and plankton. These results give evidence that more carbon was obtained through external feeding. In a previous experimental study performed on the same temperate coral, heterotrophy was found to function as an alternative source of carbon when corals were maintained in the dark, and was sufficient to maintain calcification rates at a basal value [START_REF] Hoogenboom | Co-variation between autotrophy and heterotrophy in the Mediterranean coral Cladocora caespitosa[END_REF]. The same observation, that feeding sustains carbon requirements in winter, was also made for temperate anemones and zoanthids [START_REF] Davy | Carbon budgets in temperate anthozoan-dinoflagellate symbioses[END_REF][START_REF] Bergschneider | Nutritional role of two algal symbionts in the temperate sea anemone Anthopleura elegantissima Brandt[END_REF]. Also, in winter conditions, zooxanthellae d 13 C was more depleted than in summer, showing that they might have relied to a greater extent to host-derived carbon sources.

Carbon and nitrogen signature of the different samples were simultaneously plotted in Fig. 4 in order to highlight the preferentially exploited food sources of corals. Such kinds of plots are usually used in complex food chains to determine the food source of a specific predator (Riera et al. 1999(Riera et al. , 2009)), considering a mean trophic enrichment of 1% in d 13 C [START_REF] Rau | 13 C/ 12 C and 15 N/ 14 N variations among sizefractionated marine particles: Implications for their origin and trophic relationships[END_REF]) and of 2.5-3.4% in d 15 N [START_REF] Minagawa | Stepwise enrichment of 15 N along food chains: Further evidence and the relation between d 15 N and animal age[END_REF][START_REF] Gollety | Complexity of the food web structure of Ascophyllum nodosum zone evidenced by a d 13 Ca n dd 15 Ns t u d[END_REF] for the predator as a result of the assimilation of food. It has to be noticed that the exact value of the isotopic fractionation of carbon and nitrogen between the coral host predator and its prey is not known, as it also is not known how zooxanthellae autotrophy is changing this signal. Nevertheless, application of the above model to C. caespitosa showed that the expected mean isotopic ratios for the preferentially exploited food resources (represented by an asterisk in Fig. 4) were close to the ratios of POM, SOM, and plankton in winter, and did not correspond to any of the food prey in summer. This model therefore suggests a major contribution to the host metabolism of the heterotrophic food sources in winter, and of the zooxanthellae in summer. These conclusions are corroborated by the results obtained using the model of [START_REF] Phillips | Source partitioning using stable isotopes: Coping with too many sources[END_REF], which calculated a contribution of the zooxanthellae to the host metabolism of 80-100% in summer and 0% to 20% in winter.

In conclusion, this is one of the first studies investigating, under natural conditions, the importance of auto-and heterotrophy in a temperate coral. In winter, the utilization of external food sources enables the symbiosis to persist when temperature and light conditions are unfavorable. Heterotrophy also helps in keeping a basic metabolism and some calcification when autotrophic input is not sufficient, as experimentally demonstrated in a previous study [START_REF] Hoogenboom | Co-variation between autotrophy and heterotrophy in the Mediterranean coral Cladocora caespitosa[END_REF]. Even if the photosynthetic contribution to the carbon and nitrogen budget of the coral is less important that heterotrophy during winter, there might be some aspects of the symbiosis, not measured in this study, that are beneficial to the host. Conversely, in summer, a larger autotrophic contribution and some heterotrophic feeding may help corals to increase their rates of calcification and build tissue reserves. Future work should focus on measuring the rates of photosynthesis during an annual cycle, as well as the amount and nature of metabolites translocated from the zooxanthellae to the host.

Fig. 1 .

 1 Fig. 1. Map of the three locations studied.

Fig. 2 .

 2 Fig. 2. (A) Zooxanthellae density (cells cm 22 ), (B) total Chl (mg cm 22 ), and (C) protein (mg cm 22 ) concentrations in corals sampled from Lerici, Punta Bianca, and Bonassola in winter and summer. Values are mean 6 SD of five samples.

Fig. 3 .

 3 Fig. 3. d 13 C values of zooxanthellae (zoox), host tissue (host), SOM, POM in seawater (seawater POM), and zooplankton (plankton), sampled in the three locations of Lerici, Punta Bianca, and Bonassola in winter and summer. Values are mean 6 SD of five samples.

Fig. 4 .

 4 Fig. 4. d 13 C vs. d 15 N (mean 6 SD) for the corals of the three locations, stranded sediment, zooplankton, and seawater samples for winter and summer. Asterisks correspond to the theoretical food source of the corals, taking into account the trophic enrichment of 1% and 3.5% for d 13 C and d 15 N, respectively. H, host; Z, zooxanthellae; SA, symbiotic association; PK, plankton.

  and Table 1. Within each location and each season, d 13 C of the different seawater POM fractions remained in a very close range, differing less than 1% (Table

Table 1 .

 1 ; Muller-Parker and Davy 2001; Bergschneider and Muller-Parker d 13 C mean values of the different fractions of POM and mean d 13 C values of the total POM. 1 , POM , 10, particles with a size between 1 and 10 mm; 10 , POM , 50, particles with a size between 10 and 50 mm; 50 , POM , 105, particles with a size between 50 and 105 mm; POM . 105, all particles whose size is more than 105 mm. Values in parentheses are standard deviations.

		Lerici		Punta Bianca	Bonassola
		Winter	Summer	Winter	Summer	Winter	Summer
	1,POM,10	224.8(0.5)	223.1(0.4)	224.6(0.1)	222.7(0.6)	224.3(0.5)	223.1(0.6)
	10,POM,50	225.0(0.2)	222.5(0.1)	224.4(0.2)	222.4(0.4)	224.3(0.6)	222.6(0.5)
	50,POM,105	225.6(0.7)	222.9(0.6)	224.1(0.6)	222.7(0.2)	223.7(0.7)	222.8(0.7)
	POM.105	225.7(0.7)	222.6(0.2)	224.6(0.4)	222.5(0.3)	224.2(0.3)	223.1(0.5)
	Mean	225.3(0.6)	222.8(0.4)	224.1(0.7)	222.6(0.2)	224.3(0.8)	222.8(0.4)

Table 2 .

 2 Three-way ANOVA comparing the d 13 C and d 15 N signatures between samples (host tissue, zooxanthellae, symbiotic association, POM, SOM, plankton), locations (Lerici, Punta Bianca, Bonassola), and seasons (winter, summer). p values below 0.05 show significant differences.

			d 13 C		d 15 N
	Parameter	df	FpFp
	Sample	5	120.9	,0.0001	1.3	,0.001
	Location	2	2.1	0.1290	7.4	0.001
	Season	1	317.9	,0.0001	9.3	0.003
	Sample-location	10	4.7	0.0008	1.9	0.189
	Sample-season	5	94.6	,0.0011	90.1	,0.001
	Location-season	2	3.4	0.5224	11.3	,0.001
	Sample-location-season	10	3.4	0.0069	5.2	0.042

Table 3 .

 3 d 15 N values for the different categories (host tissue, zooxanthellae, symbiotic association, POM, SOM, and plankton) in the three locations, for winter and summer.

		Winter		Summer
	Location and category	Mean	SD	Mean	SD
	Punta Bianca
	Zooxanthellae	8.80	0.45	7.32	1.02
	Host tissue	10.18	1.01	6.94	1.18
	Symbiotic association	7.93	1.12	7.02	0.04
	SOM	5.01	0.30	5.02	0.79
	POM	4.60	1.15	9.30	1.32
	Plankton	5.70	0.12	5.43	0.13
	Lerici
	Zooxanthellae	7.51	0.99	7.73	0.70
	Host tissue	8.58	1.51	8.20	0.65
	Symbiotic association	7.42	0.10	7.68	0.07
	SOM	3.15	0.19	2.48	1.09
	POM	4.15	1.27	8.40	0.86
	Plankton	5.72	0.17	5.40	0.10
	Bonassola
	Zooxanthellae	6.86	1.10	5.45	0.80
	Host tissue	7.46	1.68	5.93	0.70
	Symbiotic association	7.17	0.25	5.06	0.14
	SOM	4.51	1.09	2.27	0.20
	POM	4.74	1.83	7.29	1.24
	Plankton	5.75	0.09	5.45	0.11

important for the carbon budget of C. caespitosa in summer. This autotrophy might have been due to a lower availability of POM, SOM, and plankton during this season, or more probably to higher rates of zooxanthellae photosynthesis, and a higher contribution to the host's energy demand. High zooxanthellae productivity and photosynthesis during summer or under high light conditions has indeed been recorded for the same coral species

(Rodolfo-Metalpa et al. 2008a)

and for other temperate anthozoans (Verde and McCloskey 2007; Bergschneider

  and Haime 1849. J.Exp. Mar. Biol. Ecol. 48: 85-97, doi:10.1016/0022-0981(80)90009-X ---, AND ---. 1984. Effects of feeding frequency and symbiosis with zooxanthellae on nitrogen metabolism and respiration of the coral Astrangia danae. Mar. Biol. 81: 153-162, doi:10.1007/BF00393114 VERDE, E., AND L. R. MCCLOSKEY. 2002. A comparative analysis of the photobiology of zooxanthellae and zoochlorellae symbiotic with the temperate clonal anemone Anthopleura elegantissima (Brandt). Mar. Biol. 141: 225-239, doi:10.1007/ s00227-002-0824-7 ---, AND ---. 2007. A comparative analysis of the photobiology of zooxanthellae and zoochlorellae symbiotic with the temperate clonal anemone Anthopleura elegantissima (Brandt). III. Seasonal effects of natural light and temperature on photosynthesis and respiration. Mar. Biol. 152: 775-792, doi:10.1007/s00227-007-0737-6

Acknowledgments

We thank R. Delfanti, Director of the Marine Environment Research Centre of La Spezia, Italy; D. Allemand, Director of the Scientific Centre of Monaco; Helen Kemp and Wolfram Meier-Augenstein (James Hutton Institute, Scotland) for isotope analyses; and all those who helped us in the laboratories, and at sea collecting the corals: G. Cerrati, A. Bordone, S. Sgorbini, and R. Cupido. We also thank three anonymous reviewers who greatly improved the manuscript. Financial support was provided by funds from the Centre Scientifique de Monaco and Agenzia nazionale per le nuove tecnologie.


Marc-Arthur Loko

M.D. Ph.D Laurent Castera
email: laurent.castera@chu-bordeaux.fr

Chair François Dabis

Brigitte Le Bail

Maria Winnock

Gaëlle Coureau

Paulette Bioulac-Sage

Victor De Ledinghen

Didier Neau

Loko Marc-Arthur

Castera Laurent

Dabis François

Le Bail Brigitte

Winnock Maria

Coureau Gaëlle

Bioulac-Sage Paulette

De Ledinghen Victor

Neau Didier

The Groupe

G Chêne

M Dupon

M Longy-Boursier

P Morlat

J L Pellegrin

J M Ragnaud

Chair). R Salamon

R Thiébaut

C Lewden

S Lawson- Ayayi

P Mercié

J F Moreau

N Bernard

D Lacoste

D Malvy

M J Blaizeau

M Decoin

S Delveaux

C Hannapier

S Labarrère

V Lavignolle-Aurillac

B Uwamaliya-Nziyumvira

G Palmer

D Touchard

E Balestre

A Alioum

H Jacqmin-Gadda

M Bonarek

F Bonnet

P Gellie

K Lacombe

; M Dupon

F Dauchy

H Dutronc

S Lafarie

T Pistonne

P Thibaut

D Chambon

C De

La Taille

C Cazorla

T Galperine

A Ochoa

J F Viallard

O Caubet

E Lazaro

C Nouts

; P Couzigou

ME ; H Fleury

B Lafon

I Masquelier

D Pellegrin

J F Breilh

P Moreau

Blanco

P Dax

L Loste

Caunègre

Bayonne

F Bonnal

MC S Farbos

S Ceccaldi

Tchamgoué

S De

Validation and comparison of simple noninvasive indexes for predicting liver fibrosis in HIV-HCV-coinfected patients: ANRS CO3 Aquitaine cohort

Keywords: HIV-HCV coinfection, liver fibrosis, non-invasive indexes, platelet count

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.

INTRODUCTION

Chronic hepatitis C virus (HCV) infection is common in patients with human immunodeficiency virus (HIV) [START_REF] Alter | Epidemiology of viral hepatitis and HIV co-infection[END_REF][START_REF] Sherman | Hepatitis C Virus prevalence among patients infected with Human Immunodeficiency Virus: a cross-sectional analysis of the US adult AIDS Clinical Trials Group[END_REF]. Among the 40 million HIV-infected persons worldwide, an estimated 4-5 million are also chronically infected with HCV [START_REF] Alter | Epidemiology of viral hepatitis and HIV co-infection[END_REF]. HIV infection notably modifies the natural history of HCV infection with an accelerated progression of HCV-related liver disease towards cirrhosis [START_REF] Graham | Influence of human immunodeficiency virus infection on the course of hepatitis C virus infection: a meta-analysis[END_REF][START_REF] Martinez-Sierra | Progression of chronic hepatitis C to liver fibrosis and cirrhosis in patients coinfected with hepatitis C virus and human immunodeficiency virus[END_REF][START_REF] Martin-Carbonero | Incidence and predictors of severe liver fibrosis in human immunodeficiency virus-infected patients with chronic hepatitis C: a European collaborative study[END_REF][START_REF] Mohsen | Impact of human immunodeficiency virus (HIV) infection on the progression of liver fibrosis in hepatitis C virus infected patients[END_REF][START_REF] Salmon-Ceron | Liver disease as a major cause of death among HIV infected patients: role of hepatitis C and B viruses and alcohol[END_REF]. In addition, response to antiviral therapy in HIV-HCV coinfected is poorer than in HCV monoinfected patients with higher discontinuation rates [START_REF] Chung | Peginterferon Alfa-2a plus ribavirin versus interferon alfa-2a plus ribavirin for chronic hepatitis C in HIV-coinfected persons[END_REF][START_REF] Carrat | Pegylated interferon alfa-2b vs standard interferon alfa-2b, plus ribavirin, for chronic hepatitis C in HIV-infected patients: a randomized controlled trial[END_REF][START_REF] Torriani | Peginterferon Alfa-2a plus ribavirin for chronic hepatitis C virus infection in HIV-infected patients[END_REF]. Therefore, assessment of liver fibrosis is of critical importance in HIV-HCV coinfected patients not only for prognosis but also for antiviral therapy indications.

Until recently, liver biopsy was the only way to evaluate fibrosis [START_REF] Bravo | Liver biopsy[END_REF]. However, liver biopsy is an invasive and painful procedure with rare but potentially life-threatening complications [START_REF] Castéra | Pain experienced during percutaneous liver biopsy[END_REF][START_REF] Cadranel | Practices of liver biopsy in France: results of a prospective nationwide survey. For the Group of Epidemiology of the French Association for the Study of the Liver (AFEF)[END_REF]. Thus many patients are reluctant to undergo liver biopsies and HIV-HCV coinfected patients may be discouraged to start anti-HCV treatment for this reason. The accuracy of liver biopsy to assess fibrosis has also been questioned, in relation to sampling errors and intra-and inter-observer variability that may lead to over-or under-staging [START_REF] Bedossa | Sampling variability of liver fibrosis in chronic hepatitis C[END_REF][START_REF] Colloredo | Impact of liver biopsy size on histological evaluation of chronic viral hepatitis: the smaller the sample, the milder the disease[END_REF][START_REF] Regev | Sampling error and intraobserver variation in liver biopsy in patients with chronic HCV infection[END_REF]. These findings thus emphasize the need for accurate non-invasive methods to measure the degree of liver fibrosis.

Several markers and models have been proposed over the past few years for the prediction of fibrosis in HCV monoinfected patients, including the aspartate aminotransferase (AST) to platelet ratio index [START_REF] Wai | A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C[END_REF], the Forns index [START_REF] Forns | Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model[END_REF] and platelet count [START_REF] Lackner | Comparison and validation of simple noninvasive tests for prediction of fibrosis in chronic hepatitis C[END_REF]. However, very little information regarding the performance and utility of these tests in HIV-HCV coinfected patients is available. Finally, FIB-4 a new index based on age, alanine aminotransferase (ALT), AST and platelets has been specifically designed for predicting severe liver fibrosis in HIV-HCV coinfected patients [START_REF] Sterling | Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection[END_REF]. Although this index has been very recently studied in HCV monoinfected patients [START_REF] Vallet-Pichard | FIB-4: an inexpensive and accurate marker of fibrosis in HCV infection. comparison with liver biopsy and fibrotest[END_REF], it has not been validated independently in HIV-HCV coinfected patients.

The aim of our study was to evaluate the diagnostic performance of APRI, Forns index, FIB-4, and platelet count, for predicting liver fibrosis in HIV-HCV coinfected patients.

PATIENTS AND METHODS

Patients

This retrospective study included patients with HIV-HCV coinfection who were referred for liver biopsy prior to HCV antiviral therapy at the University Hospital of Bordeaux between January 1999 and January 2005. Inclusion criteria were: age over 18, positive serum antibodies to HCV by means of a second-or third-generation HCV enzyme-linked immunosorbent assay (Ortho Diagnostic, Raritan NJ, USA) and detectable serum HCV RNA (Amplicor TM HCV, Roche Molecular Systems, Pleasanton, California, USA). Exclusion criteria were: coinfection with hepatitis B, other known causes of liver disease, alcohol intake of more than 50g/day. A total of 200 patients of the Aquitaine Cohort met those criteria.

The ANRS CO 3 Aquitaine Cohort, a prospective hospital-based cohort of HIV-1-infected patients under routine clinical management, was initiated in 1987 in the Bordeaux University Hospital and four other public hospitals in the Aquitaine region, Southwestern France, by the Groupe d'Epidémiologie Clinique du SIDA en Aquitaine (GECSA) [START_REF] Binquet | Modeling changes in CD4-positive T-lymphocyte counts after the start of highly active antiretroviral therapy and the relation with risk of opportunistic infections: the Aquitaine Cohort, 1996-1997[END_REF]. All adult patients with HIV-1 infection confirmed by Western-Blot testing, and who have given an informed consent are enrolled in the cohort, whatever their clinical stage, gender or HIV transmission group. Additionally, information from at least one follow-up visit after the baseline assessment, or known date of death has to be available. At each hospital contact, a standardized questionnaire including epidemiological, clinical, biological and therapeutic data is filled in by clinicians and entered into the database. The schedule of follow-up visits is based on clinical practice, and an active search of patients lost to follow-up is performed annually.

Histological assessment

The liver biopsy specimen was fixed in 10% formalin, paraffin embedded, and serially sectioned. Sections were stained with hematoxylin-eosin-safran, Masson's trichrome, picrosirius red, and Gordon and Sweet's stains. Biopsy samples were read by a single experienced pathologist (BLB) who was unaware of the clinical data of the patients, using the METAVIR scoring system for fibrosis [START_REF]Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. The French METAVIR Cooperative Study Group[END_REF] : F0 = no fibrosis; F1= portal fibrosis without septa ; F2 = portal fibrosis with rare septa ; F3 = numerous septa without cirrhosis ; F4= cirrhosis.

Non invasive indexes for prediction of fibrosis

AST, ALT, gamma-GT, cholesterol and platelet count, routinely determined for all patients, were available on the day of liver biopsy for 55% of patients, and within 1 month of the time of liver biopsy for 45%. APRI, Forns index and FIB-4 were calculated using the formula originally described [START_REF] Wai | A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C[END_REF][START_REF] Forns | Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model[END_REF][START_REF] Sterling | Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection[END_REF]. The cut-offs used were those proposed in the original studies. For platelet count, a cut-off of 150 10 9 /L (19) was used for predicting cirrhosis.

Statistical analysis

Continuous variables were described by their mean and standard deviation (SD) or median and interquartile range (IQR) and categorical variables by percentages. To evaluate the diagnostic performance of the different indexes, sensitivity (Se), specificity (Spe), positive predictive value (PPV) and negative predictive value (NPV) were calculated, using cut-offs previously described for each index [START_REF] Wai | A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C[END_REF][START_REF] Forns | Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model[END_REF][START_REF] Sterling | Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection[END_REF]. The overall diagnostic performance of scores was evaluated by area under the receiver operating characteristic curves (AUROC). The percentage of patients correctly identified (PCI) was also estimated for each index. AUROCs were compared according to the procedure proposed by Hanley and McNeil (24). Statistical analyses were performed using Stata Statistical software, version 9.2 (Stata corporation, College station, TX).

8

RESULTS

Study population

The main characteristics of the 200 HIV-HCV coinfected patients included are presented in Table 1. The mean age was 39.8±6.3 years and 67% of patients were male. Their median CD4 cell counts was 500 (78-1644) cell/µl. Most patients (87%) were taking highly active antiretroviral therapy (HAART) at the time of non invasive evaluation of liver fibrosis. The mean platelet count did not differ between patients receiving HAART (n=174) or not (n=26):

191±68 vs. 171±55 10 9 /L, respectively (p=NS). Significant fibrosis (F≥2) was present in 157 patients (78.5%) and cirrhosis in 40 (20%). The mean liver biopsy length was 15.7±7.5 mm.

Biopsy length was greater than 10 mm in 154 patients (81%), and greater than 15 mm in 89 (46.8%) (Table 2).

Diagnostic value of the indexes

AUROCs were calculated to assess the overall diagnostic performance of each index for prediction of significant (F≥2), severe fibrosis (F≥3) and cirrhosis (F4) (Table 2).

AUROCs of Forns index, APRI and FIB4 for discriminating F0F1 versus F2F3F4 were 0.75, 0.77 and 0.79, respectively. AUROC of FIB-4 for discriminating F0F1F2 versus F3F4, was 0.77. AUROCs of FIB4, APRI and platelet count for discriminating F0F1F2F3 versus F4, were 0.80, 0.79 and 0.78, respectively.

Comparison of AUROC values of the different indexes did not show any statistically significant difference for both significant fibrosis and cirrhosis. Also, no difference was found according to liver biopsy length or between patients receiving HAART or not.

Indexes aimed at predicting significant fibrosis

For a FIB4 index ≤0.6, 9 of 43 patients (20.9%) without significant fibrosis at liver biopsy were correctly identified (Table 3). In addition, the presence of significant fibrosis could not be excluded as 3 out of 12 of patients with a FIB4 index ≤0.6 had significant fibrosis at liver biopsy (NPV=75%). For a FIB4 ≥1, 131 of 157 patients (83.4%) with significant fibrosis at liver biopsy were correctly identified. In addition, 131 of 151 patients with FIB4 ≥1 had significant fibrosis at liver biopsy (PPV=86.7%).

For an APRI ≤0.5, 21 of 43 patients (48.8%) without significant fibrosis at liver biopsy were correctly identified (Table 3). In addition, the presence of significant fibrosis could not be excluded with certainty, as 19 of 40 patients with APRI ≤0.5 had significant fibrosis at liver biopsy (NPV=52.5%). For an APRI ≥1.5, only 57 of 157 patients (36.3%) with significant fibrosis at liver biopsy were correctly identified. In addition, 57 of 59 patients with APRI ≥1.5 had significant fibrosis at liver biopsy (PPV=96.6%).

For a Forns index <4.2, only 34.6% of patients without significant fibrosis at liver biopsy were correctly identified (Table 3). The presence of significant fibrosis could not be excluded as 69% of patients with a Forns index <4.2 had significant fibrosis at liver biopsy (NPV=31%). For a Forns index >6.9, only 23% of patients with significant fibrosis at liver biopsy were correctly identified. In addition, all the patients with a Forns index >6.9 had significant fibrosis at liver biopsy (PPV=100%).

Overall, using FIB4, APRI and Forns index, liver biopsy could have been avoided in 70%, 39% and 25% of patients, respectively.

Index aimed at predicting severe fibrosis

For a FIB-4 ≤1.45, 90 of 129 patients (69.8%) without severe fibrosis at liver biopsy were correctly identified (Table 4). But, the presence of severe fibrosis could not be totally excluded, as 19 of 109 patients (17.4%) with FIB-4 ≤1.45 had severe fibrosis (NPV=82.6%). For a FIB-4 ≥3.25, only 22 of 71 patients (31%) with severe fibrosis at liver biopsy were correctly identified.

In addition, 22 of 31 patients with a FIB-4≥3.25 had severe fibrosis at liver biopsy (PPV=71%).

Using FIB-4, liver biopsy could have been avoided in 56% of patients.

Indexes aimed at predicting cirrhosis

For a FIB4 ≤1.45, 102 of 160 patients (63.7%) without cirrhosis at liver biopsy were correctly identified (Table 5). In addition, the presence of cirrhosis could not be excluded totally, as 7 of 109 patients with an FIB4 ≤1.45 had cirrhosis at liver biopsy (NPV=93.6%). For a FIB4 ≥3.25, 16 of 40 patients (40%) with cirrhosis at liver biopsy were correctly identified. In addition, 16 of 31 patients with an FIB4 ≥3.25 had cirrhosis at liver biopsy (PPV=51.6%).

For an APRI ≤1, 100 of 160 patients (62.5%) without cirrhosis at liver biopsy were correctly identified (Table 5). In addition, the presence of cirrhosis could not be excluded totally, as 6 of 106 patients with an APRI ≤1 had cirrhosis at liver biopsy (NPV=94.3%). For an APRI >2, 19 of 40 patients (47.5%) with cirrhosis at liver biopsy were correctly identified. In addition, 19 of 44 patients with an APRI >2 had cirrhosis at liver biopsy (PPV=43.2%).

Platelet count <150.10 9 g/l, identified correctly 27 of 40 patients (67.5%) with cirrhosis at liver biopsy (Table 5). The absence of cirrhosis could not be excluded totally as 13 of 137 (9.5%) patients with platelets count ≥150.10 9 g/l had cirrhosis at liver biopsy (NPV=90.5%).

Overall, using FIB4, APRI and platelet count, liver biopsy could have been avoided in 59%, 60% and 76% of patients, respectively.

DISCUSSION

Although non invasive markers of liver fibrosis are being increasingly used in clinical practice in HCV monoinfected patients [START_REF] Afdhal | Evaluation of liver fibrosis: a concise review[END_REF][START_REF] Castera | Evolving practices of non-invasive markers of liver fibrosis in patients with chronic hepatitis C in France: Time for new guidelines?[END_REF], data on the performance of these tests in HIV-HCV coinfected patients are lacking. The Fibrotest [START_REF] Imbert-Bismut | Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study[END_REF] was one of the few tests evaluated in HIV-HCV coinfection. In a retrospective study in 130 patients [START_REF] Myers | Serum biochemical markers accurately predict liver fibrosis in HIV and hepatitis C virus co-infected patients[END_REF], the AUROC for detection of significant fibrosis (Metavir F≥2) was 0.85. For a Fibrotest value <0.2, significant fibrosis could be excluded with 93% certainty (NPV 93%) whereas for a value >0.6, the presence of significant fibrosis could be predicted with 86% certainty (PPV 86%). Overall, liver biopsy could have been avoided in approximately 55% of patients. Recently, an index based on hyaluronic acid, albumin and AST (SHASTA) has been proposed in HIV-HCV coinfected patients [START_REF] Kelleher | Prediction of hepatic fibrosis in HIV/HCV coinfected patients using serum fibrosis markers: The SHASTA index[END_REF]. For a value <0.3, fibrosis could be excluded with 94% certainty (NPV 94%) whereas for a value >0.8 the presence of significant fibrosis could be predicted with 100% certainty. Overall, liver biopsy could have been avoided in approximately one-third of patients.

It must be stressed, however, that Fibrotest and SHASTA are based on laboratory parameters not routinely performed and use a complex formula that limit their clinical applicability.

In the present study, we assessed the diagnostic performance of several simple and inexpensive non invasive indexes based on routinely available laboratory tests (APRI, Forns index, FIB-4, and platelet count), for the prediction of liver fibrosis in HIV-HCV coinfected patients. Liver biopsy was used as the reference for the diagnosis of fibrosis. We assessed the performance of the different indexes by measuring areas under the ROC curve. Overall, areas under the ROC curves did not differ significantly between the indexes (Forns, APRI, FIB-4 or platelet count) for both significant fibrosis and cirrhosis. Also no difference was found according to liver biopsy length. Finally, the diagnostic performance of these scores was lower in HIV-HCV coinfected patients than that found in the original studies performed in HCV monoinfected patients. For instance, the diagnostic accuracy of APRI and Forns index for significant fibrosis (with AUROCs of 0.77 and 0.75, respectively) was lower than that found in the original studies [START_REF] Wai | A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C[END_REF][START_REF] Forns | Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model[END_REF] performed in HCV monoinfected patients (in which the AUROCs were 0.88 and 0.81, respectively) but similar to the latest independent reports [START_REF] Lackner | Comparison and validation of simple noninvasive tests for prediction of fibrosis in chronic hepatitis C[END_REF][START_REF] Leroy | Prospective comparison of six non-invasive scores for the diagnosis of liver fibrosis in chronic hepatitis C[END_REF][START_REF] Castera | Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C[END_REF]. APRI predicted the presence of significant fibrosis with 96.6% certainty (only 3.4% of patients with a score ≥1.5 did not have significant fibrosis), and overall 39% of patients were correctly classified.

Similarly, Forns index predicted the presence of significant fibrosis with 100% certainty. However, no more than 25% of patients could be correctly classified. Finally, liver biopsy could have been avoided in 39% and 25% of our patients using APRI and Forns index, respectively as compared with around 50% in the original studies. These findings are in line with those of Macias et al. [START_REF] Macias | Prediction of liver fibrosis in human immunodeficiency virus/hepatitis C virus coinfected patients by simple non-invasive indexes[END_REF] in 263 HIV-HCV coinfected patients in whom liver biopsy could have been avoided in only one-third of patients using APRI and Forns index for prediction of significant fibrosis.

One possible explanation for such a discrepancy between our findings and those from original studies could be the difference for the prevalence of significant fibrosis among studies: higher in our HIV-HCV coinfected population (78.5%) than in the original studies, ranging from 26 to 50% [START_REF] Wai | A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C[END_REF][START_REF] Forns | Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model[END_REF]. Also, performance of APRI and Forns index could be affected in HIV-HCV coinfected patients by factors such as HAART-associated hepatotoxicity and HIV-induced thrombocytopenia [START_REF] Cole | Ineffective platelet production in thrombocytopenic human immunodeficiency virus-infected patients[END_REF][START_REF] Kontorinis | Hepatotoxicity of antiretroviral therapy[END_REF][START_REF] Bonacini | Liver injury during highly active antiretroviral therapy: the effect of hepatitis C coinfection[END_REF]. However, in the present study, diagnostic performance (as measured by AUROCs) of Forns index and APRI as well as mean platelet count did not differ significantly between patients receiving HAART or not. On the contrary, FIB4 when used for the exclusion or the prediction of significant fibrosis (at cut-offs of 0.6 and 1, respectively) performed better than APRI and Forns index. Surprisingly, these performances (AUROC 0.79 and 70% of PCI) are better than those published in the original study (AUROC 0.71 and 52% of PCI) [START_REF] Sterling | Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection[END_REF]. We have no clear explanation for this discrepancy, except, as stated before, differences in the prevalence of significant fibrosis between the 2 studies. It should be stressed, however, that specificity was very poor (20.9% and 53.5% for cut-offs ≤0.6 and ≥1, respectively) making FIB-4 as well as APRI and Forns index currently not suitable for confident use in clinical practice in HIV-HCV coinfected patients, especially for making treatment decision.

When APRI was used for the prediction of cirrhosis, its diagnostic performance was better than that observed for significant fibrosis. For instance an APRI ≤1 could exclude cirrhosis with 94% certainty (NPV=94.3%). Similarly, FIB4≤1.45 and platelet count when ≥150 x10 9 /l could exclude cirrhosis with 94% (NPV=93.6%) and 90% certainty (NPV=90.5%).

Although this may be important for reassuring patients, it is of little clinical use as these patients still need a liver biopsy for treatment decision. By contrast, APRI, FIB4 and platelet count did not confidently predict the presence of cirrhosis. For an APRI>2, a FIB4≥3.25 and a platelet <150 x10 9 /l, the positive predictive values for cirrhosis were low (43.2%, 51.6% and 42.9%, respectively), which indicated a need of liver biopsy to stage for half of the patients. Overall, 60%, 59% and 76% of patients could be correctly classified with APRI, FIB4 and platelet count.

These results are in agreement with those obtained in HIV-HCV coinfection [START_REF] Macias | Prediction of liver fibrosis in human immunodeficiency virus/hepatitis C virus coinfected patients by simple non-invasive indexes[END_REF] as well as in HCV monoinfection [START_REF] Lackner | Comparison and validation of simple noninvasive tests for prediction of fibrosis in chronic hepatitis C[END_REF][START_REF] Leroy | Prospective comparison of six non-invasive scores for the diagnosis of liver fibrosis in chronic hepatitis C[END_REF].

With regards to the prediction of severe fibrosis (F3-F4), the diagnostic performance of FIB-4 with an AUROC of 0.77 was close to that reported in the original study [START_REF] Sterling | Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection[END_REF]. For instance, a FIB-4 ≤ 1.45 could exclude severe fibrosis with 82% certainty (NPV=82.6%).

Conversely, a FIB-4 ≥3.25 could predict the presence of severe fibrosis with 71% certainty (PPV=71.0%). However, the percentage of patients in whom liver biopsy could have been avoided (56%) was lower than in the original study (71%) [START_REF] Sterling | Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection[END_REF]. Also on a clinical standpoint, 19 patients with severe fibrosis on liver biopsy and a FIB-4 ≤ 1.45 would have been falsely reassured with the risk of inappropriate management. Such a misclassification rate remains to high for confident use of FIB-4 in clinical practice.

One way to increase diagnostic accuracy of non invasive markers in HIV-HCV coinfected patients might be to use sequential algorithms combining several markers as recently suggested in HCV monoinfected patients [START_REF] Sebastiani | Stepwise combination algorithms of non-invasive markers to diagnose significant fibrosis in chronic hepatitis C[END_REF]. Further studies are needed to validate these algorithms in HIV-HCV coinfected patients.

In conclusion, the overall diagnostic performance of these indexes was lower in HIV-HCV coinfected patients than originally reported in HCV monoinfected patients. The use of FIB-4, APRI or platelet count could avoid liver biopsy for the diagnosis of significant fibrosis, severe fibrosis and cirrhosis in up to 56 to 76 % of cases in HIV-HCV coinfected patients.

However, given the high percentage of misclassified patients, these indexes do not currently appear to be suitable for routine clinical use in HIV-HCV coinfected patients. Further external validations in larger HIV-HCV coinfected populations are still needed to optimize the use of these non-invasive methods in such patients.

What is current knowledge

-Non invasive markers of liver fibrosis are gaining popularity in HCV monoinfected patients.

-Data on the performance of these markers in HIV-HCV coinfected patients are still lacking.

What is new here -The diagnostic performance of these markers is lower in HIV-HCV coinfected patients than that originally reported in HCV monoinfected patients.

-The use of these markers could save liver biopsies in up to 56 to 76 % of cases for the prediction of severe fibrosis-cirrhosis.

-However, given the high percentage of misclassified cases for significant fibrosis, such markers do not appear currently suitable for use in clinical practice in HIV-HCV coinfected patients.

Table 1 : Demographic, biological and histological characteristics of the 200 HIV-HCV coinfected patients of the ANRS CO 3 Aquitaine cohort N=200

 1 Note: Continuous variables are expressed as mean (standard deviation); categorical variables are expressed as n (%).

	Age (years)	39.8 (6.3)
	Male gender n (%)	133 (67.0)
	Body mass index (kg/m²)	22.1 (3.2)
	Source of HIV infection n (%)
	Intravenous drug use	136 (68.0)
	Others	64 (32.0)
	AST/ULN	2.2 (1.6)
	ALT/ULN	2.3 (1.9)
	Gamma-GT a (IU/l)	154 (175.8)
	Cholesterol a (mg/dl)	171.8 (50.3)
	Platelet count (x10 9 /l)	188 (67.2)
	Liver biopsies length (mm)	15.7 (7.5)
	CD4 cell count b (/µl) n (%)
	≤200	11 (6.1)
	200-350	35 (19.6)
	>350	133 (74.3)
	HIV plasma RNA c n (%)
	Undetectable (<50copies/ml)	84 (49)
	Antiretroviral therapy n (%)	174 (87)
	PI	79 (39.5)
	NRTI	170 (85)
	NNRTI	62 (31)
	Fibrosis stage (METAVIR score) n (%)
	F0-F1	43 (21.5)
	F2	86 (43)
	F3	31 (15.5)

Table 3 . Diagnostic performance of the indexes aimed at predicting significant fibrosis (F≥2) in HIV- HCV coinfected patients.

 3

	< cut-off means absence of significant fibrosis ; >cut-off means presence of significant fibrosis

Se: sensitivity; Spe: specificity; PPV: positive predictive value; NPV: negative predictive value; PCI : patients correctly identified; AUROC: area under the receiver operating characteristic curve.

Table 4 . Diagnostic performance of the index aimed at predicting severe fibrosis (≥F3) in HIV- HCV coinfected patients.

 4 : sensitivity; Spe: specificity; PPV: positive predictive value; NPV: negative predictive value; PCI : patients correctly identified; AUROC: area under the receiver operating characteristic curve.< cut-off means absence of severe fibrosis ; >cut-off means presence of severe fibrosis

			Fibrosis stage
		All patients	F0-F2	F3-F4
	FIB-4	N =200	(N=129)	(N= 71)	Se	Spe	PPV	NPV	PCI
		n (%)	n (%)	n (%)	(%)	(%)	(%)	(%)	(%)
	≤ 1.45	109 (54.5)	90 (69.8)	19 (26.8)	73.2	69.8	57.1	82.6	56
	≥ 3.25	31 (15.5)	9 (7)	22 (31)	31	93	71	71

Se

Table 5 . Diagnostic performance of the indexes aimed at predicting cirrhosis (F4) in the HIV- HCV coinfected patients.

 5

			Fibrosis stage
		All patients	F0-F3	F4
		N =200	(N=160)	(N= 40)	Se	Spe	PPV	NPV	PCI
		n (%)	n (%)	n (%)	(%)	(%)	(%)	(%)	(%)
	FIB4 a
	≤ 1.45	109 (54.5)	102 (63.7)	7 (17.5)	82.5	63.7	36.3	93.6	59
	≥ 3.25	31 (15.5)	15 (9.4)	16 (40)	40	90.6	51.6	85.8
	APRI a
	≤ 1	106 (53)	100 (62.5)	6 (15)	85	62.5	36.2	94.3	60
	> 2	44 (22)	25 (15.6)	19 (47.5)	47.5	84.4	43.2	86.5
	Platelet b count
	< 150	63 (31.5)	36 (22.5)	27 (67.5)	67.5	77.5	42.9	90.5	76
	≥ 150	137 (68.5)	124 (77.5)	13 (32.5)

Se: sensitivity; Spe: specificity; PPV: positive predictive value; NPV: negative predictive value; PCI : patients correctly identified; AUROC: area under the receiver operating characteristic curve.

a : < cut-off means absence of cirrhosis and; >cut-off means presence of cirrhosis b : < cut-off means presence of cirrhosis and > cut-off means absence of cirrhosis