Aileen Marie Connolly

Aine Hearty

Anne Nugent

Aideen Mckevitt

Elaine Boylan

Albert Flynn

Michael J Gibney
email: mike.gibney@ucd.ie

Áine Hearty

Pattern of intake of food additives associated with hyperactivity in Irish children and teenagers

Keywords: Food additives, hyperactivity, children, teenagers, consumption 30

In 2008, the EU introduced new legislation with regards to the use of food additives 2 (Regulation (EC) Nº 1333/2008). This new legislation stipulates that any food on sale in the 3 EU which contains particular food additives (Sunset yellow, Carmoisine, Tartrazine, Ponceau 4 4R, Allura Red, and Quinoline yellow) be labelled with the words to indicate that these 5 additives "may have an adverse effect on activity and attention in children" (EC Regulation 6 2008). This change in legislation was adopted on 16 th October 2008, published on 31 st 7 October 2008 and entered into force on 20 th January 2009. It will apply from the 20 th January 8 2010. Foods entering the market or labelled before this date which do not comply may be 9 marketed until their date of minimum durability or the use-by-date. This development arose 10 from a study based in Southampton, UK, which showed that either of two mixes of food 11 additives administered to children daily over one week increased the risk of developing 12 hyperactive behaviour [START_REF] Mccann | on the effect of some colours and sodium benzoate on children's 22 behaviour[END_REF]). The study was a double-blind, randomised-13 controlled study and was subsequently analysed by two independent committees of experts, 14 the UK Committee on Toxicology (Committee on Toxicity 2007) and the European Food 15 Safety Authority (European Food Safety Authority 2007) and both fully accepted the validity 16 of the study. The present paper does not attempt to negate or contest the findings of that 17 study which clearly showed that a given hazard (either of two mixes of five food additives) 18 elicited an adverse effect (hyperactive behaviour) in a given population (children). What the 19 present study is attempting to do is to add to risk assessment data by examining the 20 probability that the hazard in question actually occurs in a nationally representative sample 21 of Irish children and teenagers. The Irish food consumption databases are particularly suited 22 to this purpose since all food intake data are collected at brand level. The collection of 23 branded data is to allow the maintenance of the Irish National Food Ingredient Database 24 INFID has many uses such as identifying which foods contain specific ingredients 1 and thus was used to establish human exposure to dioxin in the recent Irish pork dioxin 2 incident. In the case of food additive intake estimates, the great strength of INFID is its use 3 to accurately assign the true presence or absence of a target food additive in a given food. 4

Normally, in the absence of such data, the estimation of food additive intake must 5 necessarily assume that if an additive can legally be present in a food it will always be 6 present. INFID also allows for the exploration of the simultaneous ingestion of given food 7 additives whether from one food or from several foods. Thus the present paper is intended to 8 inform the risk assessment process on the pattern and level of usage of the food additives 9 used in the Southampton study. 10

Methods 11

The Irish National Food & Ingredient Database (INFID) 12

Patterns of additive intake by Irish children and teenagers were assessed using The Irish 13

National Food & Ingredient Database (INFID). INFID is a multi-faceted database developed 14

in Microsoft Access which stores information on the ingredients of 1859 foods eaten in the 15 Irish diet. This information was obtained alongside two national dietary surveys: The National 16

Children's Food Survey (NCFS), (2003 -2004) All packaging belonging to foods consumed during the survey period were collected 1 by the participants and given to the field workers conducting the surveys. This packaging 2 was then forwarded to the coordinating centre in University College Dublin. Using this 3 packaging, general information about the food was recorded in INFID, such as the brand 4 name, product description, product weight, and country of origin of the food. Ingredient lists 5 for every product were also recorded from the package label, as well as the nutritional 6 information of each product. A composite food & ingredient table also recorded information 7 on the composite ingredients in the products e.g. ingredients for the jam of a biscuit. 8

All food intake data in the surveys were collected at brand level, and unique brand 9 identification codes were assigned to separate branded food items and these differed per 10 flavour per product type. This resulted in 5551 different brand codes for the children and 11 4921 brand codes for the teenagers. Details of all ingredients per food item consumed were 12 entered into INFID and coded using unique brand id codes to link with the food consumption 13 databases. Ingredients were also identified by unique ingredient id codes, which allowed 14 INFID to be searched for a specific ingredient. For example, when INFID was explored to 15 ascertain the occurrence of Sunset Yellow (E 110) in foods consumed by the children and 16 teenagers, the ingredient table was searched for its unique ingredient code. This produced a 17 list of all the products and brand id codes that contained this food colour. 18

Food consumption data 20

Food consumption data from the NCFS and the NTFS were exported from the dietary 21 analysis software WISP © (Tinuviel Software, Anglesey, UK) and exported as food files into 22 SPSS (SPSS v.12). Each row in the food files corresponded to the weight and the nutrient 23 breakdown of each food consumed per each eating occasion per day per subject. All foods 24 consumed were also coded at brand level, thus each food was linked to a brand id code as 25

Assessing additive intake 5

Based on the assigned brand id codes per food in the food files, it was possible to establish 6 the food ingredients associated with each food eating occasion, such as food additives, 7 based on the ingredient lists in INFID. More than one additive may have been present per 8 branded food product consumed. 9

INFID was then explored to determine the probability of the intakes of different 10 combinations of seven additives in the diets of Irish children (NCFS) and teenagers (NTFS). 11

The present study focused on the seven food additives used in the Southampton study:

12 Sunset yellow (E 110), Carmoisine (E 122), Tartrazine (E 102), Ponceau 4R (E 124), Allura 13
Red (E 129), Quinoline yellow (E 104) and Sodium benzoate (E 211). 14 15

Categorisation of Foods 16

For the purpose of exposure assessment, all foods consumed by the survey participants 17 were categorized into food groups in which these seven additives are legally permitted, as 18 were put into separate food groups. For example, an iced doughnut would be categorised 1 into both the 'Fine Bakery Wares' group (doughnut) and the 'Decorations and Coatings' 2 group (icing). This resulted in 36 food groups for food colours and 34 groups for Sodium 3

benzoate. 4

The additive intake for each participant was then estimated by multiplying the 5 consumption level of each food group where the target additives were present (identified 6 from INFID) by the additive concentration based on the MPL. When the additive was absent 7 from a food the exposure was always zero. Creme software was used to handle all of the 8 datasets and to conduct the exposure assessments. (Creme Software Ltd. 2009) These 9 assessments were run by uploading information from the NCFS and the NTFS into Creme 10 software. This information included the raw food intake data from the survey diaries (on all 11 eating occasions), EU Directive food-groups and the true occurrence of the target additives 12 in those food groups. 13

The mean daily intake of each food additive for children and teenagers was used to 14 construct a distribution from which the mean and the 97.5 th percentiles of additive intakes 15 were computed. Considering each food additive separately, participants who did not 16 consume the target additive in the course of the 7-day study were excluded so that all intake 17 estimates presented are for consumers only. Analysing 'consumer only' data ensured that 18 the worst case exposure was computed, as this prevents the reduction of intake estimates 19 by the otherwise inclusion of individuals with no intake of the additive. 20

The average age of the children in this study was 8.5 years which allowed their 21 intakes to be compared to the doses given to the 7/8-year olds in the Southampton study. In 22 that study, the additive dose used for this age group was partly based on a multiple of a 23 younger group (3 years of age) because of their higher energy requirements. In the present 24 study, the average intake of energy among the teenagers (mean age = 15.4 years) was 1.2 25 times that of the children, and thus, in the present study the Southampton doses could have 26 been adjusted upwards pro rata. However, by keeping with the lower dose applicable to 8-9 1 year olds with lower energy intakes, this allowed a conservative approach to be adopted for 2 the exposure assessments to the target additives in the adolescent sample. 3 4

Results

5

Probability of Occurrence (in foods and meals) 6

By availing of the data recorded in INFID at brand level, frequencies of the target additive 7 consumption for the children and teenagers were calculated. With 594 children and 441 8 teenagers surveyed over 7 days, this resulted in 4158 child-days and 3087 teen-days. 9

Arising from these, 72024 eating occasions were consumed by the children and 46473 by 10 the teenagers. These are known as food-eating occasions. Further to this, 19795 eating 11 occasions (meals and snacks) were recorded for the children and 13541 for the teenagers. 12 These are referred to as meal-eating occasions in the present study. 13

Table 1 lists the frequency of foods eaten by Irish children and teenagers containing 14 1 or more of the target food additives. The majority of additive-containing food eating 15 occasions for both children and teenagers only contained a maximum of one of the target 16 additives (3.8% for the children, 3.1% for the teenagers). There were a total of 5551 unique 17 brand codes in the children's database and 4921 in the teenagers. Of these, the vast 18 majority did not contain any of the target additives i.e. 94.8% for the children and 96.2% for 19 the teenagers. Of those brands that contained the target additives, 279 (5%) of the children's 20 branded foods contained at least 1 of the target additives, with 194 (3.9%) for the teenagers 21 (data not shown). In the case of children, no food-eating occasion ever contained 5 or more 22 of the target additives, and in the case of teenagers no food contained any 6 of the target 23 additives. 24 A similar trend is followed when looking at additive consumption in terms of meals. 1

Table 2 presents the frequency of meal-eating occasions by Irish children and teenagers that 2 contained at least 1 of the target additives. Again, the majority of meals for both populations 3 contained at least 1 of the target additives. As the number of foods per meal eating occasion 4 decreased, the presence of an additive-containing food increased, reflecting the high 5 occurrence of additives in snack-foods eaten alone. 6 7

Occurrence per food group 8

To gain insight into the distribution of the target food additives per food group, the presence 9 of the target additives was investigated per brand per food group according to the EU 10 Directives (Table 3a, 3b and 4). Regarding food colours (Tables 3a and3b), 'Fine Bakery 11 Wares' and 'Non-Alcoholic Flavoured Drinks' were the food groups that contained the most 12 branded products consumed by both the teenagers and children. However, the food group 13 'Confectionary' recorded the most brands containing the target food colours for both the 14 teenagers and children. A number of food groups consumed by the children and teenagers 15 did not contain any of the 7 target additives. Also, not all of the EU Directive food groups 16 were consumed in the NCFS or in the NTFS. The children consumed a total of 19 food 17 groups out of the 36 compiled where the 6 target food colours were legally permitted. Of 18 these 20 food groups, 13 did not contain any of the 6 target colours. The teenagers 19 consumed 23 food groups out of the possible 36 food groups. Of these 23 food groups, 15 20 did not contain any of the 6 target food colours. Food groups, such as 'Jams, jellies, 21 A similar trend emerges when analysing the intake of Sodium benzoate (E 211) 1 (Table 4). The children consumed a total of 16 food groups out of a possible 34 food groups 2 in which Sodium benzoate is legally permitted. Of these 16 food groups, 13 did not contain 3 Sodium benzoate. The teenagers also consumed 16 food groups out of a possible 34. Of 4 these, 12 food groups did not contain Sodium benzoate. 'Non-alcoholic flavoured drinks' and 5 'non-heat treated desserts' were the food groups that contained the most brands consumed 6 by both the children and teenagers. However, while a considerable amount of the brands in 7 the 'Non-alcoholic flavoured drinks' group contained Sodium benzoate (42% of brands 8 consumed by teenagers and 40% consumed by children), no brand consumed by the 9 children in the 'Non-heat treated desserts' contained the additive. Food groups, such as 10 'Low sugar jams, jellies', 'Emulsified sauces with fat content of >60%' and 'Liquid soups and 11 broths' were regularly consumed by the children and teenagers, yet did not contain Sodium 12 benzoate. Therefore, the primary source of Sodium benzoate for both children and 13 teenagers was through the 'non-alcoholic flavoured drinks' food group. 14

Exposure to the 7 target food additives 15

Estimated mean daily and 97.5 th percentile intakes of the target additives and comparison 16 with the doses used in the Southampton study (additives in Mix A and B) are presented in 17 Table 5. In general, apart from Tartrazine, a higher proportion of children consumed the 18 target food additives compared to teenagers. This may reflect a higher preference for 19 coloured sweet confectionery in the younger age group. However, apart from Carmoisine, 20 intake of the target additives was generally higher in teenagers, which partly reflects the 21 difference in energy intakes between the two groups (8.3 MJ/d for teenagers versus 7.0 22 MJ/d for children). Among consumers of any food colour, estimated mean intakes fell below 23 the doses used in both Mixes. Also, when higher intakes were examined (i.e. at the 97.5 th 24 percentile of food additive intake), values for both children and teenagers were less than 25 those used in Mix B. In the case of Mix A, estimated intakes at the 97.5 th percentile were 26 lower for 4 food colours: Tartrazine, Ponceau 4R, Allura Red and Quinoline Yellow. In the 1 case of the preservative Sodium benzoate, the estimated 97.5 th percentile of intake for both 2 children and teenagers exceeded the dose used in both Mixes. 3

In some cases of food additive intake, it was possible that certain individual children 4 or teenagers may have had intakes greater than the doses used in either mix. For Mix B, no 5 individual child or teenager had an estimated daily intake that reached the doses used for 6 any additive except for Quinoline Yellow (n=4) and Sodium benzoate (n=53). In the case of 7

Mix A, the total number of individuals with estimated intakes exceeding the dose used were 8 15 for Sunset Yellow, 22 for Carmoisine and 53 for Sodium benzoate (Table 5). In all cases, 9 there was no child or teenager who ever achieved an intake, even at the 97.5 th percentile, on 10 all seven days at the level used in the Southampton study. The results of the present study show that these conservative estimates of intake of food 15 additives in Irish teenagers and children are below those used in the Southampton study 16 which demonstrated a relationship between food additive intake and hyperactivity in UK 17 children. The estimated upper intakes for a very small proportion of Irish teenagers and 18 children exceeded the intake levels used in the Southampton study on individual days but 19 never on seven consecutive days. The estimated intakes of additives were made assuming 20 that the additive was present at the maximum permitted level in those foods identified as 21 containing them. It is a conservative assumption which is likely to result in significant 22 overestimation of intakes. This is borne out by a recent study in Australia which found that 23 the measured concentrations of added colours in foods are mostly less than 25% of the 24 maximum permitted levels (Food Standards Australia, New Zealand 2008). 25 When analysing the food groups permitted to contain the target food additives, it is 1 clear that even though the food additives were permitted in a number of food groups, the 2 children and teenagers only consumed a small proportion of these food groups 3 (approximately half). Further to this, although the additives were permitted in these food 4 groups, the majority of the brands consumed did not contain any of the target additives. This 5 was also a contributory factor to the low exposure of the children and teenagers to the 7 6 target additives. 7

The basis for selection of the intake levels of food additives in the Southampton study 8

is not well established. The authors of that study state that the levels of additives used in Mix 9

A were based on a previous study, while those of Mix B were "selected to indicate the 10 current average daily consumption of food additives by 3-year old and 8/9-year old children 11 in the UK". The reference cited to support this statement refers to a national survey of food 12 intake in UK children (Gregory et al. 1995). This report gives details of the intake of intense 13 sweeteners but gives no data on the intake of any of the additives used in Mix A or B. 14 Moreover, there are no published data available in the literature on the intakes of these 15 additives by UK children. The authors also state that the challenge in their study was "with 16 quantities of additives equal to typical dietary intakes". The results of the present study show 17 that these doses are higher than even the highest exposures of Irish children and teenagers 18

to the target additives. The intakes of food additives in Irish children are likely to be 19 representative of those of children in the UK since food consumption patterns for Irish 20 children are comparable to those reported in the UK (Gregory et al. 200). For example, the 21 pattern of food intake for four food categories associated with the use of food colours 22 (biscuits, chocolate confectionary, non-chocolate confectionary and savoury snacks) is 23 comparable between British and Irish children (respectively, 70 and 66 g/d for intake of the 24 four food groups combined). Furthermore, although the food ingredient database used in the 25 present study is regularly updated, the usage pattern adopted for the present study was that 26 which prevailed in the retail market at the time of the Southampton study. The published paper of the Southampton study refers to the two doses of additives 1 used in terms of the intakes that would be expected from the consumption of multiple bags 2 of 56g sweets. In the case of Mix B, which was stated to equal normal exposure to the target 3 food additives in UK children; this translates into two 56g bags of sweets per day for the 3-4 year olds and 4 such bags for the 8/9-year olds. In the case of the latter, based on the 5 energy value of an international brand of coloured sweets, this is equivalent to approximately 6 900 kcal per day, and this corresponds to 53% of the average daily energy intake of UK 7 children aged 7/10 years of age. However, published data on the dietary habits of British 8 children (Gregory et al. 2000) reveals that the average contribution of all sources of sugar 9 confectionary to energy intake was just 7-9%. 10

In the present study, even more conservative deterministic estimates of food additive 11 intake were also computed (i.e. assuming that if an additive can legally be present in a food 12 category, it will always be present (data not shown)). The estimated mean intakes of all of 13 the food colours used were found to be also below those purported to be typical among 14 British children. This approach of conservatively calculating food additive intake was open to 15 the regulatory agencies which reviewed that paper of McCann et al [START_REF] Mccann | on the effect of some colours and sodium benzoate on children's 22 behaviour[END_REF]) 16 based on publicly available data but was evidently not pursued. 17 In Irish children and teenagers, levels of exposure to food additives rarely, if ever, reach the 20 levels used in the Southampton study. In addition, the concurrent consumption of the 21 combination of additives in either of the cocktails used in that study, in single foods or in 22 eating occasions of multiple foods, hardly ever occurs. These data would suggest that the 23 risk assessment process which followed from the Southampton study should be re-24 considered. 25

(

  INFID) which records all ingredients of branded foods consumed in the various food 25 consumption surveys (Gilsenan et al. 2002). 26

  and the National Teen Food Survey (NTFS), 17 (2005 -2006). The NCFS surveyed 594 children aged between 5-12 years and the NTFS 18 surveyed 441 teenagers aged between 13-17 years. Both surveys collected quantitative food 19 intake date using 7-day food diaries along with physical activity measurements, lifestyle and 20 attitude information. A detailed account of the methodology used in both surveys is available 21 elsewhere (Irish Universities Nutrition Alliance 2009). INFID is updated on a regular basis, 22 having been updated in 2004 (during the NCFS), 2007 (during the NTFS) and is currently 23 being updated as part of a new national food survey. However, when analysing the usage 24 patterns for the present study, ingredient data was used from INFID that related to the time 25 frame of the Southampton study (2007). 26

  Information on the day of the week and on the type of meal, i.e. breakfast, was 1 recorded. The NCFS food file contained 72024 rows and the NTFS food file contained 46473 2 rows of data. 3 4

  outlined in EU legislation (EU Directive No. 94/36/EC (EU Directive 1994) and EU Directive 19 No. 95/2/EC (EU Directive 1995)). These EU Directive food groups detail the Maximum 20Permitted Level (MPL) of each additive per food group (in mg/kg food). These groups were 21 developed by categorizing foods consumed in the SPSS food files according to the 22 appropriate food group outlined in the legislation. If a food comprised of more than one 23 component that may contain the additives (i.e. composite foods), the separate components 24

  marmalade', 'Sauces and seasonings' and 'Soups (dehydrated, canned, stick cubes)' are 22 examples of food groups regularly consumed by the children and teenagers, yet which did 23 not contain any of the 6 target colours.

  27

49

  European Union. 1995. EU Directive No. 95/2/EC. European Parliament and Council 1 Directive 95/2/EC of 20 February 1995 on food additives other than colours and sweeteners. 2 Official Journal of the European Communities, L61/1-53. 3 Food Standards Australia New Zealand. 2008. Survey of added colours in foods available in 5 Australia. Available from: 6 http://www.foodstandards.gov.au/_srcfiles/Colours%20Survey_Final%20Report%2022%20O 7 ct%2008%20_2_.pdf (accessed July 10, 2009) 8 Gilsenan MB, Lambe J, Gibney MJ. 2002. Irish National Food Ingredient Database: 10 application for assessing patterns of additive usage in foods. Food Addit Contam. 19(12): 11 1105-15. 12 13 Gregory JR, Collins EDI, Davies PSW, Hughes JM, Clarke PC. 1995. National Diet and 14 Nutrition Survey: children aged 1.5 to 4.5 years. Vol. 1: Report of the Diet and Nutrition 15 Survey. London: HM Stationery Office. 16 17 Gregory JR, Lowe S. 2000. National Diet and Nutrition Survey: Young people aged 4 to 18 18 years. Vol. 1: Report of the Diet and Nutrition Survey. London: HM Stationery Office. 19 20 Irish Universities Nutrition Alliance. Applied food safety and nutrition research. Available 21 from: http://www.iuna.net/index.php/research (accessed July 10, 2009)

Table 1 : Frequency of food-eating occasions containing 1 or more of the target additives Page 16 of 29 http

 1 National Teenagers Food Survey (NTFS), 2005 -2006, 13 -17 years (n = 441). **National Childrens Food Survey (NCFS), 2003 -2004, 5 -12 years (n = 594).

	* (total no.

*://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Food Additives and Contaminants

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

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Acknowledgements 2

This project was funded by the Irish Department of Agriculture, Food and Fisheries (DAFF)

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Food Additives and Contaminants

F o r P e e r R e v i e w O n l y

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Food Additives and Contaminants

F o r P e e r R e v i e w O n l y


Abderrahim ; Oussalah

Valérie M Laurent

Olivier Bruot

Jean-Louis Gueant

Denis Régent

Marc-André Bigard

Laurent Peyrin-Biroulet
email: peyrinbiroulet@gmail.com

Jean-Louis Guéant

Prof Laurent Peyrin-Biroulet

Additional benefit of procalcitonin to C-reactive protein for assessing

Keywords: Crohn's disease < Disease-based, Inflammatory bowel disease < Disease-based, Ulcerative colitis < Disease-based, Inflammation < serum procalcitonin level, Crohn's disease, ulcerative colitis, inflammatory bowel diseases

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

Procalcitonin, a prohormone of 116 amino acids is the precursor for the calcium homeostasis hormone, calcitonin, which is found in the thyroid C cells and the pulmonary endocrine cells. [START_REF] Becker | Procalcitonin in sepsis and systemic inflammation: a harmful biomarker and a therapeutic target[END_REF] It has been found to circulate at very low concentrations in normal serum and is presumably produced by the neuroendocrine cells in the thyroid gland and in the lungs. [START_REF] Becker | Procalcitonin in sepsis and systemic inflammation: a harmful biomarker and a therapeutic target[END_REF] Procalcitonin plays a major role in systemic inflammation and induces a dose-dependent increase in TNFα secretion. [START_REF] Liappis | Human leukocyte and whole blood cytokine response to exogenous procalcitonin[END_REF] In a study on blood of normal human volunteers, recombinant human procalcitonin had an inhibitory effect on leukocyte migration with marked malfunction of neutrophils that is known to occur during sepsis. [START_REF] Liappis | Human leukocyte and whole blood cytokine response to exogenous procalcitonin[END_REF] Data from experimental models demonstrated that human recombinant procalcitonin induced a dose-dependent increase in leukocyte-derived tumour necrosis factor alpha (TNFα) secretion from isolated lymphocytes. [START_REF] Liappis | Human leukocyte and whole blood cytokine response to exogenous procalcitonin[END_REF] Current evidences indicate that mediators such as TNFα, as well as interleukin (IL)-1b and IL-6, comprise the specific proximate stimuli to hyperprocalcitonaemia. [START_REF] Whang | Procalcitonin and proinflammatory cytokine interactions in sepsis[END_REF][START_REF] Redl | Possible role of TNF on procalcitonin release in a baboon model of sepsis[END_REF] TNFα is a potent stimulant of procalcitonin production and may further reinforce the procalcitonin levels in a selfperpetuating cascade fashion. [START_REF] Whang | Procalcitonin and proinflammatory cytokine interactions in sepsis[END_REF][START_REF] Redl | Possible role of TNF on procalcitonin release in a baboon model of sepsis[END_REF] Multiple studies have demonstrated that serum levels of procalcitonin are markedly increased in humans with severe infection. [START_REF] Simon | Serum procalcitonin and Creactive protein levels as markers of bacterial infection: a systematic review and metaanalysis[END_REF] Procalcitonin has also evaluated in chronic inflammatory and autoimmune conditions as a marker of disease activity. [START_REF] Moosig | Elevated procalcitonin levels in active Wegener's granulomatosis[END_REF][START_REF] Korczowski | Concentration of procalcitonin and C-reactive protein in serum and erythrocyte sedimentation rate in active autoimmune diseases in children][END_REF][START_REF] Quintana | The use of procalcitonin determinations in evaluation of systemic lupus erythematosus[END_REF] Interestingly, in patients with Wegener's granulomatosis serum procalcitonin level (SPL) was markedly elevated in patients with highly active disease in comparison with those with inactive disease. [START_REF] Moosig | Elevated procalcitonin levels in active Wegener's granulomatosis[END_REF] In children with active autoimmune processes, slight elevation of procalcitonin concentration was observed without any evidence of bacterial infection. [START_REF] Korczowski | Concentration of procalcitonin and C-reactive protein in serum and erythrocyte sedimentation rate in active autoimmune diseases in children][END_REF] In patients with inflammatory bowel diseases (IBD), the association of procalcitonin with disease activity remains poorly investigated. Two studies in IBD patients demonstrated that procalcitonin had good diagnostic value for differentiating flares of IBD from self-limited F o r P e e r R e v i e w disease activity. [START_REF] Herrlinger | Serum procalcitonin differentiates inflammatory bowel disease and self-limited colitis[END_REF][START_REF] Thia | Role of procalcitonin in infectious gastroenteritis and inflammatory bowel disease[END_REF] Only one study evaluated the diagnostic value of SPL in determining disease activity in IBD, but included a relative small number of patients with Crohn's disease (CD), which prevents to draw conclusions. [START_REF] Oruc | Diagnostic value of serum procalcitonin in determining the activity of inflammatory bowel disease[END_REF] Furthermore, none of the aforementioned studies evaluated the correlation of procalcitonin with endoscopic and radiological markers of IBD activity. [START_REF] Herrlinger | Serum procalcitonin differentiates inflammatory bowel disease and self-limited colitis[END_REF][START_REF] Thia | Role of procalcitonin in infectious gastroenteritis and inflammatory bowel disease[END_REF][START_REF] Oruc | Diagnostic value of serum procalcitonin in determining the activity of inflammatory bowel disease[END_REF] Finally, the additional diagnostic benefit of SPL evaluation to that of C-reactive protein (CRP) has never been evaluated in IBD.

The aims of this study was therefore to assess for the first time the correlation of SPL with clinical, biological, endoscopic and radiological markers of disease activity in patients with IBD and to evaluate the additional diagnostic benefit of measuring SPL to that of CRP in the assessment of disease activity in IBD.

MATERIALS AND METHODS

Study design

We performed an observational study of a single-centre cohort. Data were retrieved from a prospectively maintained database.

Population studied

All consecutive patients seen between January 15, 2008 and June 1, 2010 who had concomitant clinical, biological, and radiological evaluation with or without concomitant colonoscopy during an IBD flare or to rule out active disease were included in the study. All investigations were prescribed at the discretion of the physician. Patients were included in the analysis if an intercurrent infection was ruled out by stool analysis for enteric pathogens and Clostridium difficile toxin, and quantitative real-time polymerase chain reaction in colonic biopsies for the detection of active cytomegalovirus infection in patients with severe or

Clinical and biological markers of disease activity

Clinical disease activity scores were calculated using data collected on the day of patient's morphological evaluation: the simple clinical colitis activity index (SCCAI) [START_REF] Walmsley | A simple clinical colitis activity index[END_REF] and Truelove and Witts severity index [START_REF] Truelove | Cortisone in ulcerative colitis; final report on a therapeutic trial[END_REF] in ulcerative colitis (UC) patients, and the Crohn's Disease Activity Index (CDAI) [START_REF] Best | Rederived values of the eight coefficients of the Crohn's Disease Activity Index (CDAI)[END_REF] in CD patients. The following biological parameters were collected from electronic patients' records: serum procalcitonin, CRP, haemoglobin, hematocrit, leukocytes, platelets, albumin, and serum iron. Of note, procalcitonin is routinely used in our department to rule out superimposed infection in all hospitalized patients with IBD. SPL are measured using automated immunofluorescent assays of procalcitonin in human serum or plasma (EDTA, heparin) samples (Brahms PCT sensitive KRYPTOR kit for Brahms KRYPTOR, Hennigsdorf, Germany) according to the supplier's protocol. Normal SPL was defined as <0.05 µg/L according to supplier's reference values and normal CRP level was defined as <5 mg/L.

Endoscopy

All colonic lesions were rated according to standardized scoring systems for UC and CD as routinely used in the department. In patients with UC, the severity and extent of endoscopic lesions were assessed by the Modified Baron score. [START_REF] Feagan | Treatment of ulcerative colitis with a humanized antibody to the alpha4beta7 integrin[END_REF] Five endoscopic segments were defined: rectum, sigmoid, left colon, transverse colon, and right colon. The Modified Baron score was applied to each segment to obtain a segmental Modified Baron score. A total score was calculated from the sum of the segmental scores to obtain the total Modified Baron score. In patients with CD, the severity of endoscopic lesions was assessed by the Simplified Endoscopic Activity Score for Crohn's Disease (SES-CD). [START_REF] Daperno | Development and validation of a new, simplified endoscopic activity score for Crohn's disease: the SES-CD[END_REF] Colonoscopy was considered concomitant to MR-DWI-colonography if it was performed within 48 hours after the radiological examination, without any therapeutic intervention during this interval.

Magnetic resonance colonography

The morphological evaluation of IBD patients consisted of a magnetic resonance diffusionweighted imaging colonography (MR-DWI-colonography) without oral or rectal preparation and with or without concomitant colonoscopy as described previously. [START_REF] Oussalah | Diffusion-weighted magnetic resonance without bowel preparation for detecting colonic inflammation in inflammatory bowel disease[END_REF] Radiological evaluation of disease activity was evaluated by the 6-items Magnetic resonance score (MRscore). [START_REF] Oussalah | Diffusion-weighted magnetic resonance without bowel preparation for detecting colonic inflammation in inflammatory bowel disease[END_REF] The MR-score includes six radiological signs: 1) DWI hyperintensity, 2) rapid gadolinium enhancement after intravenous contrast medium administration, 3) differentiation between the mucosa-submucosa complex and the muscularis propria, 4) bowel wall thickening, 5) parietal oedema, and 6) the presence of ulceration(s). The definition of each radiological sign of the MR-score is provided as supplementary material (Supplemental Table 1 online). The presence and absence of a radiological sign in a given segment were rated '1' and '0', respectively. The segmental MR-score (MR-score-S) was calculated as the sum of the numerical values obtained for the six radiological signs for a given segment. The total MRscore (MR-score-T) was calculated as the sum of the MR-score-S in a patient, with values ranging from 0 to 30 in the case of UC and from 0 to 36 in the case of CD. 1 and2, respectively.

Whole IBD cohort (n=57)

Comparison of serum procalcitonin levels between active and inactive inflammatory bowel diseases

The median value of SPL was significantly higher in patients with active IBD (0.10 µg/L; IQR 25-75 th , 0.07 to 0.21) in comparison to those with inactive disease (0.07 µg/L; IQR 25-75 th , 0.06 to 0.08) (P = 0.02) (Figure 1A). Consistently, patients with CRP level >5 mg/L exhibited significantly higher SPL (0.11 µg/L; IQR 25-75 th , 0.07 to 0.21) when compared with patients with a CRP level ≤5 mg/L (0.06 µg/L; IQR 25-75th, 0.06 to 0.07) (P = 0.002) (Figure 1B).

Correlation of serum procalcitonin level with biological and radiological markers of disease activity

SPL was strongly correlated with CRP (r=0.592, P < 0.001) (Figure 2), albumin (r=-0.609, P < 0.001), haemoglobin (r=-0.500, P < 0.001), and MR-score (r=0.552, P < 0.001) and was significantly correlated with serum iron (r=-0.283; P = 0.03) and platelets (r=0.393, P = 0.003). SPL was not correlated with leukocytes (r=0.1, P = 0.5).

In the whole group of IBD patients, SPL was significantly correlated with CRP in patients with active disease (Supplemental Table 2

online). In patients with inactive IBD, SPL and

Crohn's disease

Correlation of serum procalcitonin level with clinical, biological, endoscopic and radiological markers of activity

In patients with CD (n=30), SPL was strongly correlated with all disease activity markers namely, CDAI (r=0.545, P = 0.002), CRP (r=0.626, P = 0.0002) (Figure 2), albumin (r-0.813, P < 0.0001), haemoglobin (r=-0.709, P < 0.0001), SES-CD (r=0.797, P = 0.0006), and MRscore (r=0.599, P = 0.0005) (Table 3). SPL was significantly correlated with CRP in patients with active CD as opposed to patients in remission (Supplemental Table 2

online).

Diagnostic accuracy of serum procalcitonin for detecting disease activity

In patients with CD, using ROC analysis, a SPL >0.14 µg/L demonstrated a high accuracy for detecting severe disease as defined by a CDAI ≥300 with a sensitivity of 100%, a specificity of 96%, positive and negative predictive values of 88% and 100%, respectively, and an AUROC of 0.963 (95% Confidence Interval (CI), 0.822 to 0.999; P = 0.0001) (Table 4 and Figure 3). In CD patients, SPL >0.14 µg/L had a sensitivity of 67%, a specificity of 100%, and an AUROC of 0.815 (95% CI, 0.631 to 0.932; P = 0.0005) for detecting patients with a CRP >30mg/L (Table 4). Consistently, the median SPL was significantly higher in patients with a CDAI ≥300 (0.21 µg/L; IQR 25-75 th , 0.19 to 0.30) compared with those with a CDAI <300 (0.07 µg/L; IQR 25-75 th , 0.05 to 0.07) (P = 0.0002).

In comparison with a CRP cut-off of 5 mg/L, a SPL cut-off of 0.05 µg/L had the same diagnostic accuracy (AUROC) for detecting active or severe CD (P = 0.90 and P = 0.77, respectively). However, the combination of CRP cut-off of 5 mg/L with a SPL cut-off of 0.05 5). Moreover, the diagnostic accuracy of the SPL-CRP strategy was significantly superior to that of CRP alone for diagnosing severe CD with an AUROC of 0.783 versus 0.674 (P = 0.01) (Table 5 and Figure 4).

Ulcerative colitis

Correlation of serum procalcitonin level with clinical, biological, endoscopic and radiological markers of activity

In patients with UC (n=27), SPL was correlated with the SCCAI (r=0.423, P = 0.03), CRP level (r=0.548, P = 0.003) and MR-score (r=0.536, P = 0.004). By contrast, SPL were not correlated with albumin, haemoglobin, and the total modified Baron score (Table 3). SPL was significantly correlated with CRP in patients with active UC as opposed to patients with inactive UC (Supplemental Table 2

online).

Diagnostic accuracy of serum procalcitonin for detecting disease activity

In patients with UC, ROC analysis did not reveal any significant threshold of SPL for the detection of active or severe disease (P = 0.08 and P = 0.2, respectively) (Table 6). The SPL-CRP strategy did not yield a better accuracy for diagnosing active or severe UC in comparison with CRP alone (Table 5).

DISCUSSION

This is the first study showing that SPL is correlated with clinical, biological, endoscopic and/or radiological disease activity in patients with IBD. To date three studies have evaluated procalcitonin in IBD. [START_REF] Herrlinger | Serum procalcitonin differentiates inflammatory bowel disease and self-limited colitis[END_REF][START_REF] Thia | Role of procalcitonin in infectious gastroenteritis and inflammatory bowel disease[END_REF][START_REF] Oruc | Diagnostic value of serum procalcitonin in determining the activity of inflammatory bowel disease[END_REF] By pooling UC and CD patients, we found that SPL was significantly higher in patients with active IBD. These results are in line with those of a previously reported study. [START_REF] Herrlinger | Serum procalcitonin differentiates inflammatory bowel disease and self-limited colitis[END_REF] Consistently, by pooling UC and CD patients, SPL was significantly higher in patients with a CRP >5 mg/L and was correlated with CRP levels. This finding is also in line with the study by Oruc et al. that demonstrated a significant correlation between SPL and CRP in patients with IBD. [START_REF] Oruc | Diagnostic value of serum procalcitonin in determining the activity of inflammatory bowel disease[END_REF] When considering only patients with CD, Herrlinger et al. demonstrated that SPL were significantly correlated with the CDAI, while no correlation was found with CRP level. [START_REF] Herrlinger | Serum procalcitonin differentiates inflammatory bowel disease and self-limited colitis[END_REF] By contrast, in the study by Thia et al., SPL showed no correlation with CD activity using the Harvey-Bradshaw index (HBI). [START_REF] Thia | Role of procalcitonin in infectious gastroenteritis and inflammatory bowel disease[END_REF] This discrepancy may be partly explained by the fact that CD activity has been scored with the HBI in the study by Thia et al., [START_REF] Thia | Role of procalcitonin in infectious gastroenteritis and inflammatory bowel disease[END_REF] conversely to our study and that of Herrlinger et al. [START_REF] Herrlinger | Serum procalcitonin differentiates inflammatory bowel disease and self-limited colitis[END_REF] that used the CDAI as CD activity score. In the study by Oruc et al., SPL was not correlated with CDAI. [START_REF] Oruc | Diagnostic value of serum procalcitonin in determining the activity of inflammatory bowel disease[END_REF] However, this study included only nine CD patients, which prevents any conclusion. It is noteworthy that none of the three studies [START_REF] Herrlinger | Serum procalcitonin differentiates inflammatory bowel disease and self-limited colitis[END_REF][START_REF] Thia | Role of procalcitonin in infectious gastroenteritis and inflammatory bowel disease[END_REF][START_REF] Oruc | Diagnostic value of serum procalcitonin in determining the activity of inflammatory bowel disease[END_REF] had evaluated endoscopic or radiological markers of disease activity.

Our study showed that in patients with UC, SPL was correlated with the SCCAI, CRP, and the MR-score score, whereas no correlation was found with endoscopic disease activity scores. Among the three studies that evaluated SPL in IBD patients, [START_REF] Herrlinger | Serum procalcitonin differentiates inflammatory bowel disease and self-limited colitis[END_REF][START_REF] Thia | Role of procalcitonin in infectious gastroenteritis and inflammatory bowel disease[END_REF][START_REF] Oruc | Diagnostic value of serum procalcitonin in determining the activity of inflammatory bowel disease[END_REF] only one has specifically evaluated the SPL in the subgroup of patients with UC, and did not show any correlation between SPL and clinical disease activity. [START_REF] Thia | Role of procalcitonin in infectious gastroenteritis and inflammatory bowel disease[END_REF] This difference may be partly explained by the fact that UC activity has been scored with the Physician Global Assessment 20 in the study by Thia et al., [START_REF] Thia | Role of procalcitonin in infectious gastroenteritis and inflammatory bowel disease[END_REF] conversely to our study that used the SCCAI which is a well validated disease activity index for UC. [START_REF] Higgins | The quantitative validation of non-endoscopic disease activity indices in ulcerative colitis[END_REF][START_REF] Turner | A systematic prospective comparison of noninvasive disease activity indices in ulcerative colitis[END_REF] In the study by Oruc et al. a procalcitonin cut-off value of 0.05 µg/L, found in ROC analysis, had a poor accuracy (sensitivity, 67%; specificity 42%) for detecting active IBD as defined by a CDAI >150 or a 'moderate' or 'severe' UC according to Truelove and Witts severity index. [START_REF] Truelove | Cortisone in ulcerative colitis; final report on a therapeutic trial[END_REF] Importantly, by using ROC analysis, we were able to identify a SPL cut-off of 0.14 Overall, we found that the performance of procalcitonin for evaluating disease activity was weaker in UC compared with CD. Consistently, there was no cut-off value for SPL capable of detecting severe forms of UC in our study. This is in line with previous reports. [START_REF] Herrlinger | Serum procalcitonin differentiates inflammatory bowel disease and self-limited colitis[END_REF][START_REF] Thia | Role of procalcitonin in infectious gastroenteritis and inflammatory bowel disease[END_REF] From a mechanistic point of view one can speculate that the best correlation of procalcitonin with the degree of activity of CD is linked to the fact that TNFα plays a central role in the pathogenesis of this disease.

In conclusion, our results indicate that procalcitonin might be useful in clinical practice to assess disease activity in patients with CD. Notably, we demonstrated for the first time that SPL was correlated with endoscopic and radiological markers of activity in CD, and with radiological activity in UC. In patients with CD, a threshold of 0.14 µg/L for procalcitonin may detect the most severe forms of the disease. The combination of procalcitonin with CRP would be more efficient than CRP alone for the diagnosis of active or severe CD. Our results need to be confirmed in independent studies.

F

F o r P e e r R e v i e w

Page 17 of 23

F o r P e e r R e v i e w

Page 5

 5 sur 23 steroid-refractory colitis. Our cohort of IBD patients is reported to The Commission Nationale de l'Informatique et des Libertés (N°1404720) which supervises the implementation of theJanuary 6, 1978 Act on data processing, data files, and individual liberties as amended by theAugust 6, 2004 Act relating to protection of individuals with regard to the processing of personal data.

  , 2008 and June 1, 2010, 57 patients with a diagnosis of IBD underwent clinical, biological, endoscopic, histological and/or radiologic evaluation and could be included in the analysis. The baseline characteristics of included patients with CD (n=30) or UC (n=27) are shown in Tables

  related to the small sample size (n=13).

  Page 10 sur 23 µg/L, called the 'SPL-CRP strategy', yielded a better specificity for diagnosing active or severe CD (Table

  as defined by a CDAI ≥300. Interestingly, by adding SPL measurement to routine CRP evaluation (The SPL-CRP strategy), we were able to double the specificity of the SPL-CRP strategy for diagnosing active or severe forms of CD when compared to a 'CRP alone'-based strategy.

  a A patient may receive more than one concomitant medication b Clinical evaluation of disease activity c Endoscopic evaluation of disease activity d Radiological evaluation of disease activity F o r P e e r R e v i e w Page 16 of 23

Figure 1 .

 1 FIGURES LEGENDS

Figure 2 .

 2 Figure 2. Correlation of serum procalcitonin level (µg/L) with C-reactive protein (mg/L) in

Figure 3 .

 3 Figure 3. Accuracy of serum procalcitonin level (SPL) >0.14 µg/L for detecting severe

Figure 4 .

 4 Figure 4. Diagnostic accuracy of serum procalcitonin level >0.05 µg/L, C-reactive protein >5

Figure 4 .

 4 Figure 1a. Oussalah et al.

Table 1 . Baseline characteristics of patients in the Crohn's disease group (n=30)

 1

	o r
	P
	e
	e r
	R
	e
	v i e
	w

a A patient may receive more than one concomitant medication b Clinical evaluation of disease activity c Endoscopic evaluation of disease activity d Radiological evaluation of disease activity

Table 2 . Baseline characteristics of patients in the ulcerative colitis group (n=27)

 2

	Ulcerative colitis (n=27)	n	%
	Ulcerative colitis topography according to Montreal classification
	E1 (Ulcerative proctitis)	1	4
	E2 (Left sided ulcerative colitis)	7	26
	E3 (Extensive ulcerative colitis)	19	70
	Concomitant medication(s) a
	Mesalamine	10	37
	Oral or intravenous corticosteroids	10	37
	Azathioprine	7	26
	Methotrexate	0	-
	Cyclosporine	4	15
	Tumour Necrosis Factor antagonists	6	22
	Male gender	14	52
	Tobacco use	2	7
	Previous abdominal IBD-related surgery	0	-
		Mean Standard deviation
	Patient age at inclusion (years)	37	14
	Haemoglobin (g/dL)	11.3	2.1
	Hematocrit (g/dL)	34.0	5.9
	Leukocytes (x 10 9 /L)	9.200	3.708
	Platelets (x 10 9 /L)	430	151
	Albumin (g/L) (n=24)	32.3	8.0
	Simple clinical colitis activity index b	7	4
	Total modified Baron score c (n=15)	8	5
	Magnetic-Resonance score (0-30) d	18	8
		Median	IQR 25-75 th
	Patient age at IBD diagnosis (years)	28	23 to 36
	Disease duration at inclusion (months)	58	19 to 127
	C-reactive protein (mg/L)	14.2	6.0 to 38.0
	Serum iron (mg/L)	0.33	0.21 to 0.64

NOTE. IBD: Inflammatory bowel diseases; IQR: Interquartile range 25-75 th percentile.

Table 3 . Correlation of serum procalcitonin concentration (µg/L) with clinical, biological, endoscopic and radiological markers of disease activity in patients with inflammatory bowel disease

 3

			CD group (n=30)			UC group (n=27)
		rho a	95% CI	P-value a rho a	95% CI	P-value a
	Clinical marker of disease activity
	Crohn's disease activity index	0.545 0.230 to 0.757	0.002	-	-	-
	Simple clinical colitis activity index	-	-	-	0.423 0.051 to 0.692	0.03
	Biological markers of disease activity
	C-reactive protein (mg/L)	0.626 0.343 to 0.805	0.0002	0.548 0.212 to 0.768	0.003
	Albumin (g/L)	-0.813 -0.928 to -0.557 < 0.0001 -0.333 -0.649 to 0.081	0.1
	Haemoglobin (g/dL)	-0.709 -0.852 to -0.469 < 0.0001 -0.137 -0.492 to 0.256	0.5
	Endoscopic markers of disease activity
	SES-CD	0.797 0.462 to 0.933	0.0006	-	-	-
	Total modified Baron score	-	-	-	0.454 -0.076 to 0.784	0.09
	Radiological marker of disease activity
	MR-score	0.599 0.304 to 0.789	0.0005	0.536 0.195 to 0.761	0.004

NOTE. SES-CD: Simplified Endoscopic Activity Score for Crohn's Disease; MR-score: Magnetic-Resonance score. a Spearman's rho rank correlation coefficient.

Table 4 . Diagnostic accuracy of serum procalcitonin (µg/L) according to 'a priori' defined cut-offs for disease activity in patients with Crohn's disease P-value a AUROC a Standard Error a ROC-defined procalcitonin cut-off (µg/L) a

 4

	Sensitivity	Specificity	PPV	NPV
	(%)	(%)	(%)	(%)

NOTE. AUROC: Area under the receiver operating characteristic curve; ROC: Receiver operating characteristic; PPV: Positive predictive value; NPV: Negative predictive value. a According to DeLong, et al. 19 Page 18 of 23

Table 5 . Diagnostic accuracy of serum procalcitonin level >0.05 µg/L, C-reactive protein >5 mg/L, and their combination for detecting active or severe Crohn's disease and ulcerative colitis

 5 Comparison of AUROCs according to Delong et al.[START_REF] Delong | Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach[END_REF]

		CRP a	SPL b	CRP a and SPL b	CRP a and SPL b versus CRP a
			Active Crohn's disease	P-value c
	AUROC	0.574	0.597	0.716
	Standard Error	0.102	0.101	0.0963	0.14
	95% CI, AUROC 0.381 to 0.752 0.403 to 0.771 0.523 to 0.865
	Sensitivity (%)	77	82	68
	Specificity (%)	38	38	75
	PPV (%)	77	78	88
	NPV (%)	38	43	46
			Severe Crohn's disease	P-value c
	AUROC	0.674	0.652	0.783
	Standard Error	0.0508	0.0491	0.0528	0.01
	95% CI, AUROC 0.479 to 0.833 0.457 to 0.816 0.595 to 0.911
	Sensitivity (%)	100	100	100
	Specificity (%)	35	30	57
	PPV (%)	32	30	41
	NPV (%)	100	100	100
			Active ulcerative colitis	P-value c
	AUROC	0.877	0.545	0.855
	Standard Error	0.103	0.0314	0.105	0.32
	95% CI, AUROC 0.694 to 0.971 0.344 to 0.737 0.666 to 0.960
	Sensitivity (%)	96	9	91
	Specificity (%)	80	100	80
	PPV (%)	96	100	95
	NPV (%)	80	20	67
			Severe ulcerative colitis	P-value c
	AUROC	0.692	0.503	0.657
	Standard Error	0.0702	0.0525	0.0788	0.32
	95% CI, AUROC 0.486 to 0.854 0.305 to 0.699 0.450 to 0.827
	Sensitivity (%)	100	93	93
	Specificity (%)	39	8	39
	PPV (%)	64	52	62
	NPV (%)	100	50	83
	NOTE. AUROC: Area under the receiver operating characteristic curve; CI: Confidence
	interval; PPV: Positive predictive value; NPV: Negative predictive value.
	a C-reactive protein >5 mg/L

b Serum procalcitonin level >0.05 µg/L c

Table 6 . Diagnostic accuracy of serum procalcitonin (µg/L) according to 'a priori' defined cut-offs for disease activity in patients with ulcerative colitis P-value a AUROC a Standard Error a ROC-defined procalcitonin

 6

		Sensitivity	Specificity	PPV	NPV
	cut-off (µg/L) a	(%)	(%)	(%)	(%)

a According to DeLong, et al.

[START_REF] Delong | Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach[END_REF]

b 'Mild', 'moderate' or 'severe' ulcerative colitis according to Truelove and Witts severity index

[START_REF] Truelove | Cortisone in ulcerative colitis; final report on a therapeutic trial[END_REF]

and a SCCAI 18 >5 c According to Truelove and Witts severity index

[START_REF] Truelove | Cortisone in ulcerative colitis; final report on a therapeutic trial[END_REF]

Table 1 . Definition of the 6 components of the Magnetic Resonance score

 1

	Radiological item of the Magnetic resonance score	Plane	MR sequence	Definition
	Diffusion-weighted imaging hyperintensity	Axial	Diffusion-weighted imaging	Presence of hyperintensity in the colonic wall segment in the DWI sequence.
	Rapid gadolinium enhancement after intravenous contrast medium administration	Axial	Gradient-echo T1-weighted, arterial phase	Gadolinium enhancement in the arterial phase (20 to 25 seconds after gadolinium infusion)
	Differentiation between the mucosa-submucosa complex and the muscularis propria	Axial	Single shot fast spin echo short time echo without fat saturation	Distinction in the colonic wall between two layers: 1) mucosa-submucosa complex hyperintensity; 2) muscularis propria hypointensity.
	Bowel wall thickening	Axial and coronal	Gradient-echo T1-weighted, delayed phase	Thickness of the colonic wall exceeding 5 mm.
	Parietal edema	Axial and coronal	Single shot fast spin echo, short time echo, without fat saturation	Thickness of the colonic wall exceeding 5 mm and hyperintensity of the mucosa-submucosa complex.
			2D Steady-state, free precession imaging
	Ulceration	Axial and coronal	(Fast Imaging Employing Steady-state Acquisition, General Electric Company,	Loss of substance in the mucosa-submucosa complex.
			US)

F o r P e e r R e v i

e w Supplemental Table 2. Correlation between serum procalcitonin concentration (µg/L) and C-reactive protein (mg/L) in each subgroup of patients according to inflammatory bowel disease type and activity Active disease a Inactive disease Sample size rho b 95% CI, rho P-value b Sample size rho b 95% CI, rho P-value b

  CI: confidence interval; IBD: Inflammatory bowel diseases; CD: Crohn's disease; UC: Ulcerative colitis. Active Crohn's disease: Crohn's disease activity index ≥ 150; Active ulcerative colitis: 'mild', 'moderate' or 'severe' UC according to Truelove and Witts severity index[START_REF] Truelove | Cortisone in ulcerative colitis; final report on a therapeutic trial[END_REF] and a SCCAI 18 >5

	IBD	44	0.613 0.386 to 0.770 < 0.0001	13	-0.329 -0.745 to 0.271	0.27
	CD	22	0.704 0.401 to 0.868 0.0003	8	-0.378 -0.855 to 0.445	0.36
	UC	22	0.514 0.118 to 0.769	0.02	5	-0.316 -0.937 to 0.785	0.60
	NOTE.

a b Spearman's coefficient of rank correlation (rho)

ACKNOWLEDGEMENTS

1. Authors' declaration of personal interests: AO, VL, OB, JLG, DR, MAB, and LPB declare no conflict of interest.

2. Declaration of funding interests: This work did not receive any financial support.

Definition of active and severe disease

In order to achieve a pooled analysis, IBD patients were considered to have an active disease if they had a CDAI ≥150 in case of CD and in case of UC if they had 'mild', 'moderate' or 'severe' UC according to Truelove and Witts severity index [START_REF] Truelove | Cortisone in ulcerative colitis; final report on a therapeutic trial[END_REF] , and a SCCAI >5. [START_REF] Jowett | Defining relapse of ulcerative colitis using a symptom-based activity index[END_REF] Patients were considered to have severe disease if they have a CDAI ≥300 or a 'severe' UC according to Truelove and Witts severity index. [START_REF] Truelove | Cortisone in ulcerative colitis; final report on a therapeutic trial[END_REF]

Statistical analyses

Quantitative variables are described as means and standard deviation (S.D.) or as medians and percentiles (I.Q.R. for Inter Quartile Range: 25-75 th ) in the case of an abnormal distribution.

Proportions are expressed as percentages and 95% confidence intervals. All correlations were studied using Spearman's nonparametric correlation coefficients. The diagnostic accuracy of serum procalcitonin (µg/L) for detecting severe forms of IBD was evaluated using receiver operating characteristic (ROC) analysis according to DeLong et al [START_REF] Delong | Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach[END_REF] to calculate the sensitivity, specificity, positive, and negative predictive values, and area under the receiver operating characteristic curve (AUROC) with the associated P-value. The comparison of AUROCs was carried out using the procedure proposed by Delong et al. [START_REF] Delong | Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach[END_REF] The comparison of serum procalcitonin values between different groups of disease activity was performed using the Kruskal-Wallis test. All the reported P-values were two-sided, and P-values of <0.05 were considered statistically significant. Statistical analyses were performed using MedCalc software, version 11.3.3.0 (MedCalc Software, Mariakerke, Belgium).


J Fatome

B Kibler

M El-Amraoui

J-C Jules

G Gadret

F Desevedavy

F Smektala

Mid-infrared extension of supercontinuum in a chalcogenide suspended core fibre through soliton gas pumping

We experimentally demonstrate the mid-infrared extension of supercontinuum in a 1.9-µm suspended core chalcogenide fibre whose zero dispersion wavelength is shifted to 1.9 µm, i.e., into the transparency window of standard silica fibres. Based on the well-known long-pulse regime of supercontinuum generation in silica fibre, we are able to convert and shift a low-cost optical quasi-CW source at 1.53 µm into a large number of interacting femtosecond solitons localized up to 2.1 µm. This optical source called a soliton gas allows us to pump a 50-cm long chalcogenide microstructured fibre near its zero dispersion wavelength, thus leading to an efficient extension of a mid-infrared supercontinuum beyond 2.4 µm.

Introduction: Fibre-based supercontinuum (SC) sources in the mid-infrared (mid-IR) have a great potential for many applications such as optical frequency metrology, optical tomography and spectroscopy. It is well known that soft-glass fibres are promising candidates for such devices due to their low mid-IR losses and high intrinsic nonlinearities in opposition to silica [START_REF] Dudley | Supercontinuum generation in optical fiber[END_REF]. However, the zero dispersion wavelengths (ZDW) of these materials are generally beyond 2 µm and far from the wavelengths of conventional fibrebased laser sources. Consequently, the use of non-silica fibres often requires costly and high power pump lasers to achieve an efficient spectral broadening up to the mid-IR.

During the last decade, research activities have been focused on the development of microstructured optical fibre (MOF) technology for dispersion-tailored waveguides as well as shifted near-infrared lasers to longer wavelengths so as to pump the fibre under test close to its ZDW [START_REF] Dudley | Supercontinuum generation in optical fiber[END_REF]. In this Letter, we propose to combine these both attractive options by using the Raman soliton self-frequency-shift process (SSFS) occurring around 1550 nm in a highly nonlinear silica fiber (HNLF) combined with a chalcogenide suspended core fiber which ZDW has been shifted from 4.8 µm to 1.9 µm through its MOF profile in order to extend a SC in the mid-IR region beyond 2.4 µm.

Experimental configuration:

The experimental set-up is sketched in Fig. 1 and is basically made of two stages cascaded SC generation towards the mid-IR. The first stage shifts the laser frequency through Raman SSFS which is commonly used to shift ultrafast 1550-nm pulses to lower frequencies in silica fibers [START_REF] Xia | Mid-infrared supercontinuum generation to 4.5µm in ZBLAN fluoride fibers by nanosecond diode pumping[END_REF][START_REF] Hagen | Generation of a continuum extending to the midinfrared by pumping ZBLAN fiber with an ultrafast 1550-nm source[END_REF]. To this end, we use a low-cost and extremely compact commercial high-power sub-nanosecond pump. Indeed, our passively Q-switched micro-laser delivers 3.7 ns pulses at 1535 nm, with an average power around 19 mW and a pulse repetition rate of 2.7 kHz, corresponding to pulse energy of 7.5 μJ and peak power up to 2 kW. Based on the well-known long-pulse regime of SC generation in silica fibre [START_REF] Dudley | Supercontinuum generation in photonic crystal fiber[END_REF], we take advantage both of modulation instability (MI) and Raman SSFS processes to convert our quasi-continuous-wave (CW) source at 1.53 µm into a large number of interacting femtosecond solitons around 2 µm. The fibre is a 50-m long segment of highly nonlinear silica fibre (HNLF) characterized by a chromatic dispersion parameter D = 0.6 ps/km/nm and a nonlinear Kerr coefficient γ = 10 W -1 .km -1 at the laser frequency. The fibre exhibits a low dispersion slope D S = 0.007 ps/km/nm 2 and losses around 0.6 dB/km at 1.55 µm. By using a simple 20x microscope objective (MO) we reach about 40% of coupling efficiency into the HNLF. In this configuration, the SC formation begins with the MI of the continuous pump wave in the anomalous dispersion region, thus evolving into a train of ultra-short soliton pulses [START_REF] Dudley | Supercontinuum generation in photonic crystal fiber[END_REF][START_REF] Travers | Blue solitary waves from infrared continuous wave pumping of optical fibers[END_REF]. Since MI is a noisy seeded process, the generated solitons will have variable bandwidths and energies.

Based on the fibre parameters and the input pump power, we calculate the maximum MI gain at a frequency Ω max = 22.6 THz [START_REF]Nonlinear Fiber Optics[END_REF]. In the time domain, the CW is thus converted into a periodic pulse train characterized by a period T = 2π / Ω max = 45 fs. The soliton pulses therefore exhibit a temporal width below the period value. Their bandwidth is large enough for intra-pulse Raman scattering to occur leading to a continuous red-shift along the HNLF. Moreover, the different peak powers create a large range of self-frequency shifts, creating a smooth red-shifted continuum [START_REF] Travers | Blue solitary waves from infrared continuous wave pumping of optical fibers[END_REF][START_REF] Xia | Supercontinuum generation in silica fibers by amplified nanosecond laser diode pulses[END_REF] (see also next section). This specific spreading in time and frequency domains of the large number of interacting solitons has been already related to the idea of the soliton gas [START_REF] Travers | Blue solitary waves from infrared continuous wave pumping of optical fibers[END_REF]. The frequency shift of the soliton gas is a function of the HNLF length used and it can be suitably adjusted in order to pump the chalcogenide MOF as close as possible to the ZDW, as shown in the next section.

Note that the long wavelength extent of the HNLF continuum will be limited here by the high losses of silica glass beyond 2.1 µm.

In order to extend the SC in the mid-IR, the second stage of our setup (see Fig. 1) consists in injecting the resulting HNLF soliton gas into a 50-cm long segment of chalcogenide MOF designed with a suspended core made of As 2 S 3 glass. The input signal was coupled into the chalcogenide fiber by means of the alignment option of an optical fiber splicing device combined with a 3-µm waist micro-lens fibre, which allows an efficient fiber coupling up to 30-40% [START_REF] El-Amraoui | Strong infrared spectral broadening in low-loss As-S chalcogenide suspended core microstructured optical fibers[END_REF].

Figure 2(a) shows the corresponding SEM image of the fibre cross section. Here, the main idea is to use the attractive dispersive and nonlinear characteristics of this small core MOF with a diameter close to 1.9 µm. The calculated dispersion curve of the suspended core MOF is presented in Fig. 2(b) and the ZDW is predicted at wavelengths close to 1.9 µm by taking into account 10% discrepancies on the fibre core size. From our mode calculations, we have also deduced an effective mode area about 2.4 µm 2 at the ZDW, corresponding to an estimated nonlinear Kerr coefficient γ = 3.86 W -1 .m -1 with n 2 = 2.8 10 -18 m 2 /W [START_REF] El-Amraoui | Strong infrared spectral broadening in low-loss As-S chalcogenide suspended core microstructured optical fibers[END_REF]. Recently, we have also reported in a chalcogenide MOF that Raman gain is 180 larger than fused silica [START_REF] Fatome | Linear and nonlinear characterizations of chalcogenide photonic crystal fibers[END_REF], which underlines the potential of this kind of fiber combined with anomalous dispersion for the SSFS process. We thus expect that pumping our suspended core MOF with the soliton gas source in the anomalous dispersion regime will efficiently extend the SC towards the mid-IR by using very short lengths of fibre. Note that our chalcogenide fibre exhibits a large transparency bandwidth in the mid-IR with typical losses about 0.5 dB/m measured in the wavelength range studied [START_REF] El-Amraoui | Strong infrared spectral broadening in low-loss As-S chalcogenide suspended core microstructured optical fibers[END_REF]. However, extrinsic losses due to impurity absorption (i.e., OH-absorption) affect the glass transmission which can result in high loss absorption peaks at particular wavelengths such as 1.4 and 1.9 µm, see Fig. 3 and experimental results section.

Results and discussion: The silica and chalcogenide fibers are then chosen and arranged with the final aim to achieve the most significant spectral broadening towards the mid-IR.

Figure 3(a) first depicts the experimental results recorded at the output of the HNLF fiber for 4 different lengths of fiber. As expected, the MI of the continuous pump wave evolving into ultrashort soliton pulses induces intra-pulse Raman self-frequency shifts and subsequently a smooth SC generation. As the length of the HNLF increases, we clearly observe an accumulation of variable bandwidths and energies pulses at the leading edge of the SC qualified as soliton gas [START_REF] Travers | Blue solitary waves from infrared continuous wave pumping of optical fibers[END_REF]. By carefully adjusting the length of the HNLF between 20 and 50 m, we can then localize this soliton gas around 2 µm in a range of 200 nm. Beyond 50 m, the induced SSFS of the soliton gas reaches the transparency limit of the silica fiber, leading to a rapid decrease of its available energy. Note that the high-frequency part of the SC observed in the HNLF below 1.4 µm in Fig. 3 is generated through dispersive wave formation in the normal dispersion regime. Indeed, the MIinduced solitons are spectrally broad enough such that they overlap with the normal dispersion region of the HNLF (ZDW ~ 1.4 µm), and can excite dispersive waves at phase-matched wavelengths [START_REF] Xia | Mid-infrared supercontinuum generation to 4.5µm in ZBLAN fluoride fibers by nanosecond diode pumping[END_REF][START_REF] Hagen | Generation of a continuum extending to the midinfrared by pumping ZBLAN fiber with an ultrafast 1550-nm source[END_REF].

When now pumping the 50-cm long chalcogenide MOF with the different soliton gas described above (Fig. 3(b)), we clearly observe that the SSFS phenomenon starts again so as to extend the initial SC beyond 2.4 µm (limit of our OSA), which proves that the chalcogenide fiber is pumped into its anomalous dispersion regime. Note that for the 10-m long HNLF configuration, the residual energy contained inside the 1535-nm nanosecond pump is then sufficiently high to generate two spontaneous Raman stokes components in the chalcogenide MOF (Fig. 3(b), lightest grey solid line). Finally, the optimum SC extension in the chalcogenide MOF beyond 2.4 µm was obtained for a HNLF length of 30 m and an associated soliton gas localized around 2 µm, which could be interpreted as a ZDW below 2 µm for the Chalcogenide fiber. Indeed, this is confirmed by the fact that the SC begins its extension by pumping with the soliton gas localized over the range 1.8-2 µm (10-m-long segment of HNLF).

Conclusions:

In conclusion, we have shown that it is possible to extend a silica-based supercontinuum beyond 2.4 µm through the generation of a soliton gas inside a HNLF and its injection beyond the ZDW in a 50-cm long chalcogenide suspended core MOF.

These results reveal that the concatenation of suitably designed optical fibers, in particular based on different materials, could be an attractive solution in order to generate and extend SC sources in the mid-IR region and especially far beyond the silica transparency limit.

Figure Captions: Fig. 1

 1 Figure Captions:

Fig. 2 (

 2 Fig.2(a) SEM image of the cross section of the chalcogenide microstructured fibre

Fig. 3 (

 3 Fig.3(a) Generation of the soliton gas at the output of the HNLF as a function of fiber

FIGURE 1 Fig. 1

 11 FIGURE 1

FIGURE 2 Fig. 2

 22 FIGURE 2

FIGURE 3 Fig. 3

 33 FIGURE 3

Acknowledgements: This research was supported by the Agence Nationale de la Recherche (FENOPTIC ANR-09-NANO-023-04 and Confian project) and the Conseil Regional de Bourgogne. We thank Yokogawa and Absys for equipment support. We also thank I. Skripatchev, C. Plocchini, and Y. Messaddeq (Institute of Chemistry, Araraquara, Brazil) for their help on the drawing process.