Jonas F Ludvigsson
email: jonasludvigsson@yahoo.com

David S Sanders

Markus Maeurer

Jerker Jonsson

Johan Grunewald

Jan Wahlström

Jerker Jonsson∫

Johan Grunewald∞

Jan Wahlström∞

Risk of tuberculosis in a large sample of patients with coeliac disease -A nationwide cohort study

Keywords: Coeliac disease < Disease-based, Epidemiology < Topics, Small intestine < Organ-based, X Coeliac disease, CI, Confidence Interval, HR, Hazard Ratio, Mtb, Mycobacterium Tuberculosis, TB, Tuberculosis, VA, Villous atrophy Tables: 3. Figures: 1

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Authors: This analysis was not an afterthought but at the same time not one of our main analyses. It was performed in order to look at temporal relationships between CD and TB, since our main analyses concerned CD and subsequent risk of TB.

When recalculating our data we have noted an error in the conditional logistic regression. This error was due to us listing exclusions of individuals (Figure 1) in the wrong order (some controls with data irregularities (the n=247) were listed both in the second "level" and then in the fourth "level" of the flowchart). This did not affect the overall number of individuals in the main analysis (the main analysis of this paper is still based on 29,026 individuals with CD and 144,160 matched controls and risk of subsequent TB), but did affect the logistic regression.

We have recalculated our data and the OR for future CD is 1.6 and not 2.0 as first presented in our manuscript. We have attached the statistical output for this analysis below for the editors and peer reviewers to see.

LOGISTIC REGRESSION (COELIAC DISEASE: Risk of future CD in patients with TB)

As can be seen in figure 1 in the manuscript the number of individuals with TB agrees with that of the output above. We have extended the methods section and the results section with regards to the analysis of prior TB and future CD.

In the methods section we have listed absolute numbers, type of matching, and how we defined TB. In the results section we have added proportions, and the median duration from TB diagnosis

Case-control study (Methods)

In a case-control study we used conditional logistic regression to examine the relationship between prior TB and subsequent CD. TB was defined according to relevant ICD codes (Appendix). This analysis was based on 29,096 individuals with CD and 144,522 controls matched for sex, age, calendar period and county. We then looked at the number individuals with a prior diagnosis of TB in each group.

Risk of subsequent CD in patients with prior TB

Individuals with TB were at a 1.6-fold increased risk of having a later diagnosis of CD (95% CI odds ratio=1.2-2.1). Out of 29,096 individuals with CD, 69 (0.24%) had an earlier diagnosis of TB vs. 209/144,522 (0.15%). The median duration from TB to CD diagnosis was 10 years.

Reviewer: -While employing multiple definitions of TB demonstrates the robustness of the results, it would be ideal to show that this definition of TB is internally valid, by, for example, calculating the risk of TB among those with known risk factors. Country birth was used as a covariate in this paper, and so the authors could consider calculating and reporting the risk of TB among those with a non-nordic country of birth as compared to those with a nordic country of birth. (In the US, foreign birth is one of the strongest risk factors for developing TB.) Authors: The reason why we did not do this initially was because the number of study participants born outside the Nordic countries was small in our study. There were only 944 individuals with CD born outside the Nordic countries and 7 of these developed TB. This corresponded to a HR of 3.6 (95%CI=0.9-14.4; i.e. of borderline statistical significance p=0.072). Since individuals born outside the Nordic countries only made up 3.2% of all participants this risk increase had little effect of the summary estimate of our study. Among the 28,078 individuals born in the Nordic countries who had CD, the HR for TB was 2.1; 95%CI=1.3-3.4. As can be noted, the HR in this subset of individuals was higher than the summary estimate (2.0; 1.3-3.0). This because in this stratified analysis, a number of reference individuals from Non-Nordic countries were excluded and this drove up the risk estimates (to 2.1). Strata were not matched for nationality.

In the revised manuscript, we have added the following lines (italicised): Moreover, results were similar when we adjusted for country of birth (HR=2.2; 95% CI=1.4-3.3), or restricted our analysis to individuals born in the Nordic countries (HR=2.1; 95%CI=1. 3-3.4).

Reviewer: -It would be of interest to report the HR of developing TB among those with inflammation and those with positive serologies and negative biopsy, compared to controls. (We can infer that these groups have an increased risk of TB compared to the general population as their risk of TB was similar to the CD group.) Given the increased mortality rates reported in these groups by Dr. Ludvigsson previously, especially among those with inflammation, further analysis of morbidity in these groups would be valuable. Authors: Below is the output of the main analysis, now including individual estimates for "inflammation" (bpsgr0123(2)) and normal mucosa but positive CD" (bpsgr0123(1)) as requested by the peer reviewer. (bpsgr0123(3)) = Coeliac disease We have added the following text to our manuscript:

Methods

In a second post-hoc analysis we also estimated the risk of TB, defined as having a relevant ICD-code (Appendix) in individuals with either inflammation (Marsh 1-2) or normal mucosa (Marsh 0) and positive CD serology compared to that in age-and sex-matched reference individuals.

Results

In a second post-hoc analysis we also estimated the risk of TB in individuals with either inflammation without VA or normal mucosa but positive CD serology compared to that in age-and sex-matched reference individuals. The risk of TB was increased in both groups, although only in inflammation (HR=1.9; 95%CI=1.2-3.1) was the risk increase statistically significant (normal mucosa but positive CD serology: HR=1.8; 95%CI=0. 7-4.6). ***** Please note that HRs seem highest (2.0) in patients with CD (inflammation 1.9; normal mucosa but positive CD serology: 1.8). That the internal comparison found that patients with CD were at a slightly lower risk of TB than other individuals undergoing biopsy is due to different age-sex-and calendar period-distribution among patients undergoing biopsy (for this we refer to our paper in JAMA). For the convenience of the peer reviewer we have added Table 1 Still, the difference was not statistically significant when tested. We have discussed this specifically with Editor Professor Kane who suggested us to add that had we had access to a larger dataset we might have been able to show a difference according to sex. We have added the following lines to the revised manuscript:

Although the HR for TB in men with CD (2.9) was higher than in women with CD (1.5), this difference was not statistically significant. However, we cannot rule out that in a larger dataset than ours, men may with CD be at a higher risk of TB than women with CD.

Reviewer: You should also comment, in the discussion, that the number of observed/expected cases is very small and may not be a big public health issue, as it may appear. Other health problems, in the celiacs, are more prevalent. Reviewer: Anyway TB cases have to be screened for CD: this is important. Authors: We agree and have added the following sentences to the discussion:

Still we admit that the number of observed/expected cases of TB is limited in this study, and consequently absolute risks are low. Other health problems than TB may be of greater concern for the CD community. Nevertheless, the increased ORs for CD in individuals who first had a diagnosis of TB signal that screening for CD may be considered in patients with TB and gastrointestinal symptoms. The positive association between CD and TB was also observed before the CD diagnosis (odds ratio=1.6; 95% CI=1.2-2.1).

Conclusions

We found a moderately increased risk of TB in patients with CD.

Abstract word count: 227

Keywords: autoimmunity, coeliac, inflammation, tuberculosis individuals, resulting in chronic inflammation of the small intestine. Many patients suffer from extraintestinal manifestations, such as type 1 diabetes 2 , reproductive disorders [START_REF] Rostami | Coeliac disease and reproductive disorders: a neglected association[END_REF] , irritable bowel syndrome 4 and sepsis [START_REF] Ludvigsson | Coeliac disease and risk of sepsis[END_REF] .

Mycobacterium tuberculosis (TB) infects about one third of the world's population, although active TB is less common 6 . TB can affect any part of the body but most often occurs in the lungs, where it causes tissue destruction.

Several studies and case reports have indicated a positive association between CD and TB [START_REF] Williams | Coeliac syndrome due to tuberculous enteritis[END_REF][START_REF] Hindle | Significance of a Flat Small-Intestinal Mucosa[END_REF][START_REF] Barry | A case of small-intestinal mucosal atrophy[END_REF][START_REF] Fung | Malabsorption and subtotal villous atrophy secondary to pulmonary and intestinal tuberculosis[END_REF][START_REF] Williams | Susceptibility to tuberculosis in patients with coeliac disease[END_REF][START_REF] Peters | Causes of death in patients with celiac disease in a population-based Swedish cohort[END_REF] . In 2007, our research group found that patients with a CD inpatient diagnosis were at a 4-fold increased risk of developing TB in the future. However, that study may have overestimated the risk of TB in CD since individuals with CD were identified using the Swedish inpatient register and not all individuals with CD require hospital admission. Furthermore, in that study [START_REF] Ludvigsson | Coeliac disease and risk of tuberculosis: a population based cohort study[END_REF] we were not able to confirm TB against external data.

In this study we examined the association between biopsy-verified CD and TB. In subanalyses we also calculated the risk of TB (1) with a record of TB medication, (2) confirmed through the Swedish national surveillance system for infectious diseases or (3) through positive cultivation. Finally, we explored whether the increased risk of TB in CD was restricted to individuals with villous atrophy (VA) or independent of small intestinal histopathology.

METHODS

Through the personal identity number [START_REF] Ludvigsson | The Swedish personal identity number: possibilities and pitfalls in healthcare and medical research[END_REF] , we linked nationwide data on small intestinal histopathology with data on Swedish national TB registers. We defined CD as having a record of VA at small intestinal biopsy (see Table A1 in the appendix.).

Tuberculosis was defined according to relevant international classification of disease (ICD) codes, in the Swedish outpatient and inpatient registers (appendix).

Collection of biopsy data

We collected data from duodenal/jejunal biopsy reports with VA (Marsh stage 3 and equal to CD) [START_REF] Ludvigsson | Validation study of villous atrophy and small intestinal inflammation in Swedish biopsy registers[END_REF] from all pathology departments in Sweden (n=28). Data collection took place between October 2006 and February 2008 while the biopsies had been performed between 1969 and 2008 (Table 1). Each individual undergoing biopsy was matched with up to five reference individuals by age, sex, county and calendar year through the Total Population Register.

For comparative reasons (secondary reference groups), we also collected data on patients with inflammation without VA (Marsh 1-2) and normal mucosa but positive CD serology (Marsh 0 with positive IgA/IgG for antigliadin, endomysial (EMA) or tissue transglutaminase (TTG)) [START_REF] Ludvigsson | Symptoms and signs in individuals with serology positive for celiac disease but normal mucosa[END_REF] . More information on that data collection has been published in two validation papers [START_REF] Ludvigsson | Validation study of villous atrophy and small intestinal inflammation in Swedish biopsy registers[END_REF][START_REF] Ludvigsson | Symptoms and signs in individuals with serology positive for celiac disease but normal mucosa[END_REF] .

When examining TB in patients with CD, we used the same dataset as in our paper on overall mortality in CD [START_REF] Ludvigsson | Smallintestinal histopathology and mortality risk in celiac disease[END_REF] . This dataset consisted of 29,096 individuals with CD.

Additional exclusions among patients with CD and their reference individuals are shown in Figure 1.

Statistics

We used internal stratified Cox regression to estimate Hazard ratios (HRs) for TB.

The internal stratification means that the index individual was only compared with his or her reference individuals within the same stratum and then a summary risk estimate was calculated. The proportional hazards assumption was tested through plotting log minus log curves. Follow-up started on the date of the first biopsy with VA and the corresponding date in matched reference individuals. Follow-up ended with TB diagnosis, death, emigration or on Dec 31, 2008, whichever happened first.

In a priori subanalyses we examined the risk of TB by follow-up period, sex, age and calendar year at CD diagnosis. We calculated incidence rates as the number of first recorded TB diagnoses divided by person-years at risk.

In separate analyses we adjusted for type 1 diabetes mellitus, education and country of birth (Nordic country vs. non-Nordic country). In a post-hoc analysis we examined the risk of TB in individuals with CD compared with the risk in secondary reference groups. In a second post-hoc analysis we also estimated the risk of TB, defined as

Case-control study

In a case-control study we used conditional logistic regression to examine the relationship between prior TB and subsequent CD. TB was defined according to relevant ICD codes (Appendix). This analysis was based on 29,096 individuals with CD and 144,522 controls matched for sex, age, calendar period and county. We then looked at the number individuals with a prior diagnosis of TB in each group.

We used SPSS 18.0 to perform all analyses. HRs with 95% CIs that did not include 1 were regarded as statistically significant. The majority of the study participants were females (Table 1). The median age was 30 years at study entry with 40% undergoing biopsy in childhood. Most individuals were biopsied after 1990 (Table 1).

Coeliac disease and subsequent TB

During follow-up, 31 individuals with CD and 74 reference individuals developed TB. The HR for TB was 2.0 (95% CI=1.3-3.0). HRs decreased with follow-up (Table 2). When we excluded the first year of follow-up, the risk of TB was 1.7 (95% CI=1.1-2.8).

The overall HR for TB in CD was 1.5 in women (95% CI=0.8-2.7) and 2.9 in men (95% CI=1.6-5.3)(Table 3). The difference between women and men with CD was not statistically significant (p=0.104).

TB was uncommon in children and only in middle-aged and elderly people was the association between CD and TB statistically significant (Table 3). The age differences in HRs for TB, however, were only of borderline significance (p=0.058).

Linkage with other registers

The Swedish Prescribed Drug Register was initiated in mid-2005 and therefore covered only a very small part of the study period. In our study five individuals with CD had a TB diagnosis and a record of TB medication (reference individuals, n=10), corresponding to a HR of 2.9 (95% CI=1.0-8.3).

Of the individuals with a TB diagnosis in either the inpatient or outpatient register, the diagnosis could be confirmed in the TB register of the National Institute for Infectious Disease Control in Sweden in 11 patients with CD (reference individuals, n=18). Thus, the HR for having a TB diagnosis confirmed in the national surveillance system was 2.6 (95% CI=1.3-5.2). Of the 11 patients with CD and TB, 8 suffered from pulmonary TB (72%), which can be compared with 15/18 reference individuals (83%) (Fisher's exact test, p=0.646).

The HR increased to 3.3 (95% CI=1.6-6.8) when we restricted our outcome to a TB diagnosis confirmed by positive TB cultivation according to the Swedish Institute for Infectious Disease Control.

Post-hoc analyses

Individuals with CD (VA) were at no increased risk of TB when compared with individuals with inflammation without VA (adjusted HR=0.9; 95% CI=0.5-1.6) or those with normal mucosa but positive CD serology (adjusted HR=0.7; 95% CI=0.3-1.9).

In a second post-hoc analysis we also estimated the risk of TB in individuals with either inflammation without VA or normal mucosa but positive CD serology compared to that in age-and sex-matched reference individuals. The risk of TB was increased in both groups, although only in inflammation (HR=1.9; 95%CI=1.2-3.1) was the risk increase statistically significant (normal mucosa but positive CD serology: HR=1.8; 95%CI=0.7-4.6).

Risk of subsequent CD in patients with prior TB

Main findings and comparison with previous research

Several studies have examined the relationship between CD and TB [START_REF] Williams | Coeliac syndrome due to tuberculous enteritis[END_REF][START_REF] Hindle | Significance of a Flat Small-Intestinal Mucosa[END_REF][START_REF] Barry | A case of small-intestinal mucosal atrophy[END_REF][START_REF] Fung | Malabsorption and subtotal villous atrophy secondary to pulmonary and intestinal tuberculosis[END_REF][START_REF] Williams | Susceptibility to tuberculosis in patients with coeliac disease[END_REF][START_REF] Peters | Causes of death in patients with celiac disease in a population-based Swedish cohort[END_REF][START_REF] Ludvigsson | Coeliac disease and risk of tuberculosis: a population based cohort study[END_REF][START_REF] Stevens | Lung cavities in patients with coeliac disease[END_REF] . Two of these studies calculated risk estimates among patients with CD [START_REF] Peters | Causes of death in patients with celiac disease in a population-based Swedish cohort[END_REF][START_REF] Ludvigsson | Coeliac disease and risk of tuberculosis: a population based cohort study[END_REF] with rather high relative risks for both incident TB (HR=3.74; 95% CI=2.14-6.53) [START_REF] Ludvigsson | Coeliac disease and risk of tuberculosis: a population based cohort study[END_REF] and death from TB (standardized mortality ratio, SMR=5.9; 95% CI=1.6-15.1) [START_REF] Peters | Causes of death in patients with celiac disease in a population-based Swedish cohort[END_REF] .

The slightly lower risk of TB in this study (HR=2.0) as compared with that in the two above-mentioned studies [START_REF] Peters | Causes of death in patients with celiac disease in a population-based Swedish cohort[END_REF][START_REF] Ludvigsson | Coeliac disease and risk of tuberculosis: a population based cohort study[END_REF] can probably be explained by our non-selected group of patients. We included patients with biopsy-verified CD throughout Sweden, whereas the two previous studies were restricted to inpatients with CD [START_REF] Peters | Causes of death in patients with celiac disease in a population-based Swedish cohort[END_REF][START_REF] Ludvigsson | Coeliac disease and risk of tuberculosis: a population based cohort study[END_REF] . Inpatients with CD are likely to have a more intense disease activity with more complications (including TB and other diseases) than the average patient with CD. That surveillance bias plays an important role for the association is clear from the higher risk estimates in the first year after biopsy when the HR for TB was 3.5. In contrast, the risk of TB more than five years after diagnostic biopsy with VA was only 1.6 and did not reach statistical significance. The lower risk estimate for TB in our study cannot be explained by lack of statistical power. In the study by Peters et al [START_REF] Peters | Causes of death in patients with celiac disease in a population-based Swedish cohort[END_REF] the expected number of deaths from TB was only 0.7; in our study on incident TB published in 2007 (Ludvigsson et al, 2007) we expected to find 6.4 cases with TB [START_REF] Ludvigsson | Coeliac disease and risk of tuberculosis: a population based cohort study[END_REF] . This should be compared with 16 expected cases of TB in our current study. Still we admit that the number of observed/expected cases of TB is limited in this study, and consequently absolute risks are low. Other health problems than TB may be of greater concern for the CD community. Nevertheless, the increased ORs for CD in individuals who first had a diagnosis of TB signal that screening for CD may be considered in patients with TB and gastrointestinal symptoms.

Whereas Peters et al did not separate children and adults in their study on mortality in CD al [START_REF] Peters | Causes of death in patients with celiac disease in a population-based Swedish cohort[END_REF] , our previous study [START_REF] Ludvigsson | Coeliac disease and risk of tuberculosis: a population based cohort study[END_REF] found increased risks of TB in both children (HR=3.44) and adults (HR=3.84). This finding contrasts with the lack of association between CD and TB in patients diagnosed 0-19 years of age or 20-39 years of age in the current study. However, we cannot rule out the risk of type 2 errors (and that we have failed to identify a true risk increase) in children since stratified analyses had lower power than the all-inclusive main analysis and since the interaction between CD and age on the risk of TB was non-significant.

Although the HR for TB in men with CD (2.9) was higher than in women with CD (1.5), this difference was not statistically significant. However, we cannot rule out that in a larger dataset than ours, men with CD may be at a higher risk of TB than women with CD.

Strengths and limitations

We used biopsy reports with VA from all Swedish pathology departments (n=28) to identify CD. Biopsy reports have a high sensitivity for diagnosed CD [START_REF] Ludvigsson | Validation study of villous atrophy and small intestinal inflammation in Swedish biopsy registers[END_REF] . In a questionnaire study 15 96% of Swedish adult gastroenterologists and 100% of pediatric gastroenterologists reported performing small intestinal biopsy before CD diagnosis in at least 9 out of 10 patients. On average, 3 biopsies are taken [START_REF] Ludvigsson | Symptoms and signs in individuals with serology positive for celiac disease but normal mucosa[END_REF] , which is likely to rule in about 95% of patients with CD [START_REF] Pais | How many duodenal biopsy specimens are required to make a diagnosis of celiac disease?[END_REF] . We also reviewed patient charts and laboratory data in 114 randomly selected patients in our cohort and concluded that 108 (95%) of those with VA had CD [START_REF] Ludvigsson | Validation study of villous atrophy and small intestinal inflammation in Swedish biopsy registers[END_REF] . Furthermore, we reviewed a large number of biopsy reports (through computerized searches followed by manual review). This review found that other diagnoses than CD were very uncommon in individuals with VA (any inflammatory bowel disease, 0.3%; Helicobacter pylori, 0.2% and gastric metaplasia, 0.1%) [START_REF] Ludvigsson | Validation study of villous atrophy and small intestinal inflammation in Swedish biopsy registers[END_REF] .

In validated patients with VA and available data on CD serology 88% had positive CD serology prior to biopsy. We did, however, not require positive CD serology for the diagnosis of CD since a study based on CD serology would have diminished our follow-up since CD serology was only introduced in routine health care in the late 1990s. In contrast, VA has been in use since the 1970s and has remained critical for the CD diagnosis ever since. The long follow-up and large number of study participants increased statistical power and allowed for important subanalyses.

In our main analysis we used both the inpatient and the outpatient registers to identify patients with TB. To our knowledge, the TB diagnosis has not yet been validated in any of these registers. Based on validations of other diseases, we expect a positive predictive value of about 80-90% [START_REF] Linnersjo | Recent time trends in acute myocardial infarction in Stockholm, Sweden[END_REF][START_REF] Michaelsson | Hormone replacement therapy and risk of hip fracture: population based case-control study. The Swedish Hip Fracture Study Group[END_REF] . It is important to note that HRs for TB increased when we increased the specificity through validation against independent TB data from (1) the Swedish Prescribed Drug Register, (2) the Swedish national surveillance system for infectious disease and (3) TB cultivation data from the five Swedish national TB laboratories.

We did not adjust for smoking data. Smoking is a risk factor for TB, but often inversely associated with CD. Accordingly, lack of smoking data is not likely to explain our findings. This assumption is supported by data from the current study on women with CD and at least one record in the Swedish Medical Birth Register where smoking is recorded. Of 3,902 women with CD, 18.3% indicated that they were smokers, which can be compared with 20.8% of 18,987 reference women.

Mechanisms

There may be several mechanisms, not mutually exclusive, that could explain the association between CD and tuberculosis. One potential explanation could be vitamin D deficiency. CD may result in malabsorption of vitamin D and gluten-free products are often low in vitamin D content. Low serum levels of vitamin D are associated with increased risk of active tuberculosis, as shown by a recent meta-analysis of previous studies [START_REF] Nnoaham | Low serum vitamin D levels and tuberculosis: a systematic review and meta-analysis[END_REF] . Support for an association between vitamin D and TB is also provided by findings that genetic variants of the vitamin D receptor have been linked to the risk of TB infection [START_REF] Liu | VDR and NRAMP1 gene polymorphisms in susceptibility to pulmonary tuberculosis among the Chinese Han population: a case-control study[END_REF] . Vitamin D has been found to promote the antimicrobial activity of the innate immune system, which provides a rapid response against invading pathogens. For example, vitamin D was shown to induce the synthesis of nitric oxide in macrophages, resulting in suppression of M. tuberculosis (Mtb) [START_REF] Bettelli | Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells[END_REF] . The discovery of human IL-17+ Foxp3+ T-cells underlines the common nature of these cells [START_REF] Voo | Identification of IL-17-producing FOXP3+ regulatory T cells in humans[END_REF] . Autoimmunity appears to be driven, in part, by IL-17 immune responses and overt IL-17-positive immune responses may, depending on the frequency of exposure to mycobacterial species, be detrimental in the orchestrated immune response to Mtb. [START_REF] Cruz | Pathological role of interleukin 17 in mice subjected to repeated BCG vaccination after infection with Mycobacterium tuberculosis[END_REF] . Thus, the nature of Tregs along with IL-17-producing cells may shape an environment facilitating CD (increased Th17 However, another study found evidence for reduced Treg function in CD [START_REF] Granzotto | Regulatory T-cell function is impaired in celiac disease[END_REF] , and even if Treg cells are increased in CD, they do not seem to prevent CD from being associated with many autoimmune diseases.

Co-infection or 'co-disease' may lead to a chronic inflammatory process with a concomitant increased CTLA-4 and PD-1 expression on immune cells, which is associated with decreased Th17 and Th1 responses. Blockage of PD-1 restored IFNgamma responses and blockage of CTLA-4 restored both Th1 and Th17 responses [START_REF] Babu | Human type 1 and 17 responses in latent tuberculosis are modulated by coincident filarial infection through cytotoxic T lymphocyte antigen-4 and programmed death-1[END_REF] .

We could hypothesize that CD represents such a 'chronic inflammatory situation', which may be associated with reduced immune responsiveness (defined by increased PD-1 and CTLA-4 expression on T-cells). This situation may facilitate Mtb infection because of the reduction of, in particular, Th1 responses, which are crucial to contain or fight off Mtb.

Finally, one possible way in which bacterial infection (such as TB) could promote the development of CD is via the induction of fever since that could lead to the increased transport of gluten-derived peptides across the intestinal epithelium, which, in turn, enhances the sensitization to gluten peptides [START_REF] Bethune | Parallels between pathogens and gluten peptides in celiac sprue[END_REF] . Even at physiologically relevant temperature increases, the permeability of intestinal epithelium is increased [START_REF] Dokladny | Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability[END_REF] .

We cannot rule out that some individuals with TB were identified as part of the investigations for CD. The highest risk estimates were seen in the first year after diagnosis even if the HR for TB remained statistically significant thereafter.

Compared with individuals undergoing small intestinal biopsy but not having VA, individuals with CD (with VA) were at no increased risk of TB. This observation suggests that severe histopathological changes may not be a prerequisite for later TB in patients with CD.

In conclusion, we found a positive association between CD and TB, both before and after diagnosis of CD. This finding suggests shared risk factors or an increased risk of TB in patients with undiagnosed CD. Education. We used seven predefined levels, ranging from not completed the 9-year compulsory school to the PhD-degree. This information was obtained from the Education Register.

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

Country of birth. This information was obtained from the government agency Statistics Sweden and we used the customary division (Nordic countries vs. the rest of the world).

  . Ludvigsson et al. Risk of tuberculosis in a large sample of patients with coeliac disease 2 CD."

  . Ludvigsson et al. Risk of tuberculosis in a large sample of patients with coeliac disease 3 to CD (10 years).

  . Ludvigsson et al. Risk of tuberculosis in a large sample of patients with coeliac disease 4

3 AbstractBackground

 3 Research suggests a positive association between coeliac disease (CD) and tuberculosis (TB), but that research has often been limited to inpatients and small sample size. We examined the relationship between TB and CD.MethodsWe collected biopsy data from all pathology departments in Sweden (n=28) to identify individuals who were diagnosed with CD between 1969 and 2007 (Marsh 3: villous atrophy; n=29,026 unique individuals). Population-based sex-and agematched controls were selected from the Total Population Register. Using Cox regression, we calculated hazard ratios (HRs) for TB from data in the Swedish national health registers. Results Individuals with CD were at increased risk of TB (HR=2.0; 95% CI=1.3-3.0) (during follow-up, 31 individuals with CD and 74 reference individuals had a diagnosis of TB). The absolute risk of TB in patients with CD was 10/100,000 person-years with an excess risk of 5/100,000. Risk estimates were highest in the first year. Restricting our outcome to a diagnosis of TB confirmed by (I) a record of TB medication (HR=2.9; 95% CI=1.0-8.3), (II) data in the National Surveillance System for Infectious Diseases in Sweden (HR=2.6; 95% CI=1.3-5.2) or (III) positive TB cultivation (HR=3.3; 95% CI=1.6-6.8) increased risk estimates.

INTRODUCTION

  Coeliac disease (CD) is an immune-mediated disease occurring in 1% of the Western population[START_REF] Dube | The prevalence of celiac disease in average-risk and at-risk Western European populations: a systematic review[END_REF] . The disease is triggered by gluten exposure in HLA-DQ2+ and DQ8+

  OutcomeOur main outcome was a diagnosis of TB (appendix). In subanalyses we restricted our outcome to individuals with a TB diagnosis confirmed by a second independent data source: (1) a record in the Swedish national surveillance system for infectious diseases (appendix), (2) a record of TB medication according to relevant ATC codes (J04A) in the Swedish Prescribed Drug Register (appendix) and (3) positive TB cultivation in one of Sweden's five TB labs specialized in TB.

  ICD-code (Appendix) in individuals with either inflammation (Marsh 1-2) or normal mucosa (Marsh 0) and positive CD serology compared to that in age-and sex-matched reference individuals.

  This study included 29,026 individuals with CD and 144,160 general population reference individuals. Secondary reference groups consisted of 13,261 individuals with inflammation and 3710 individuals with normal mucosa but positive CD serology.

  1 diabetes (HR=2.1; 95% C=1.4-3.2) or education (HR=2.1; 95% C=1.3-3.4) did not change the risk estimates. Moreover, results were similar when we adjusted for country of birth (HR=2.2; 95% CI=1.4-3.3), or restricted our analysis to individuals born in the Nordic countries (HR=2.1; 95%CI=1.3-3.4).

  Individuals with TB were at a 1.6-fold increased risk of having a later diagnosis of CD (95% CI odds ratio=1.2-2.1). Out of 29,096 individuals with CD, 69 (0.24%) had an earlier diagnosis of TB vs. 209/144,522 (0.15%). The median duration from TB to CD diagnosis was 10 years.

DISCUSSION

  This nationwide population-based study found a 2-fold increased risk of TB in patients with biopsy-verified CD. This increased risk in TB was highest in the first year of follow-up. Children with CD were at no increased risk of TB. A positive association between CD and TB was also noted before CD diagnosis.

24 .

 24 Other mechanisms have recently been elucidated, including the role of vitamin D together with IL-15 to mediate the effect of pathogen-induced stimulation of the pattern-recognition receptor TLR2 to induce expression of the antimicrobial peptide cathelicidin in macrophages 25 . Furthermore, increased frequency of Foxp3+ CD25+ regulatory T-cells has been demonstrated in the blood of CD patients 26 . An increased Treg frequency could dampen the response of effector T-cells needed to combat M. tuberculosis with a subsequent higher risk to develop clinical TB. Increased numbers of Foxp3+ CD25+ Treg cells, capable of suppressing Mtb-specific T-cell cytokine production, have been demonstrated in the blood from patients with active TB 27 . The clinical relevance of Treg represents an intense field of research and the cytokine Interleukin-17 (IL-17A) may be instrumental in deciding the fine balance between protective immune responses and autoimmunity. TGFβ and Il-6 differentiate precursor T-cells to IL-17producing Th17 cells, TGFβ alone induces Treg cells and both cells have common stages in their development

  response and Tregs) as well as clinical active TB; both of these conditions are characterized by increased Treg frequency and Th17-associated immune responses[START_REF] Monteleone | Characterization of IL-17A-producing cells in celiac disease mucosa[END_REF]

  in a large sample of patients with coeliac disease -A nationwide cohort study Jonas F Ludvigsson, David S Sanders, Markus Maeurer, Jerker Jonsson, Johan Grunewald, Jan Wahlström Definition of Tuberculosis Individuals with a relevant ICD code (ICD-7: 001-019; ICD-8: 010-018; ICD-9: 010-019; ICD-10: A15-19) in the Swedish inpatient or outpatient register were defined as suffering from TB. The inpatient register began in 1964, covering all Swedish counties since 1987. The outpatient register records all hospital outpatient visits since 2001. Description of registers used in this paper 1. Swedish National Surveillance System for Infectious Diseases. This register contains data on TB between 1989 and 2007 and reporting is mandatory for both clinical and laboratory physicians. There are five TB laboratories in Sweden.2. The Swedish Prescribed Drug Register was established in mid-2005 and initially covered 84% of all drugs used in Sweden. This register, however, does not cover use of drugs in the wards.

  mellitus. Defined according to relevant ICD codes. In ICD 7, 8 and 9 there was no distinction between type 1 and type 2 diabetes. In this study we defined type 1 diabetes as having an inpatient diagnosis of diabetes before age 30 years.

  from that paper.

	0926-2010. Ludvigsson et al. 0926-2010. Ludvigsson et al.
	Risk of tuberculosis in a large sample of patients with coeliac disease Risk of tuberculosis in a large sample of patients with coeliac disease
	F o r F o r
	P P
	e e
	e r e r
	R R
	e e
	v i e v i e
	w w
	6

Table 1

 1 Characteristics of study participants Positive coeliac disease serology 180 days before biopsy and until 30 days after biopsy. †Follow-up time until diagnosis of TB, death from other cause, emigration or Dec 31, 2008. In reference individuals follow-up can end through small intestinal biopsy. ‡ Sweden, Denmark. Finland, Norway and Iceland.

	18

*

TABLE 2 .

 2 Risk of TB based on follow-up time

	Follow-up Observed	Expected	HR;	P-value	Absolute risk/	Excess risk/	Attributable
		events	events	95% CI		100,000 PYAR	100,000 PYAR	percentage
	All	31	16	2.0; 1.3-3.0	0.001	10	5	50
	Year <1	8	2	3.5; 1.5-8.4	0.005	28	20	72
	1-4.99	10	5	1.9; 0.9-3.8	0.082	10	5	47
	5+	13	8	1.6; 0.8-3.1	0.164	8	3	37
	PYAR, Person-years at risk.
	Reference is general population comparator cohort

e e r R e v i e w 22

TABLE 3 .

 3 Risk of TB (subgroup analyses).

	Subgroup Observed	Expected	HR;	P-value	Absolute risk/	Excess risk/	Attributable
		events	events	95% CI		100,000 PYAR	100,000 PYAR	percentage
	Sex
	Males	17	6	2.9; 1.6-5.3	0.001	15	10	65
	Females	14	10	1.5; 0.8-2.6	0.205	8	2	32
	Age
	<20 yrs	1	2	0.4; 0.1-3.2	0.399	1	-	-
	20-39 yrs 3	2	1.3; 0.4-4.4	0.701	6	1	21
	40-59 yrs 11	4	2.7; 1.3-5.4	0.007	16	10	62
	60+ yrs	16	6	2.5; 1.4-4.8	0.004	40	24	61
	Calendar
	period
	-1989	5	4	1.2; 0.4-3.3	0.719	6	1	17
	1990-1999 17	7	2.4; 1.3-4.3	0.004	11	7	58
	2000-	9	4	2.1; 1.0-4.5	0.048	14	7	53
	PYAR, Person-years at risk.
	Reference is general population comparator cohort

Alimentary Pharmacology & Therapeutic

This project (2006/633-31/4) was approved by the Research Ethics Committee of the Karolinska Institute, Sweden on June 14 th , 2006.

Writing Assistance: None.

Grant Support (Funding)

JFL was supported by a grant from the Örebro University Hospital while writing this article.

This project was supported by grants from The Swedish Society of Medicine, the Swedish Research Council -Medicine (522-2A09-195), the Sven Jerring Foundation, the Örebro Society of Medicine, the Karolinska Institutet, the Clas Groschinsky Foundation, the Juhlin Foundation, the Majblomman Foundation, Uppsala-Örebro Regional Research Council and the Swedish Coeliac Society. Independence (role of the sponsors): None of the funders had any role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript.

data in the study and takes responsibility for the integrity of the data and the accuracy of the data analyses.

Conflicts of interest/Disclosure requirement

All authors declare that they have no conflicts of interest and therefore nothing to declare

Ethics

The study was approved by the Research Ethics Committee of Karolinska Institutet.

Additional details on the method are provided in the appendix.


MD, PhD Laurent Peyrin-Biroulet
email: peyrinbiroulet@gmail.com

MD Abderrahim Oussalah

MD Xavier Roblin

MD Miles P Sparrow

Prof Laurent Peyrin-Biroulet

Use of azathioprine during pregnancy and in the postoperative setting in

Keywords: Thiopurines < Topics, Inflammatory bowel disease < Diseasebased, Crohn's disease < Disease-based, Pregnancy / women's issues azathioprine, mercaptopurine, thiopurines, inflammatory bowel diseases (IBD), Crohn's disease, postoperative recurrence, pregnancy, survey

Use of azathioprine during pregnancy and in the postoperative setting in Crohn's disease: A worldwide survey of experts

INTRODUCTION

Over the last two decades, treatment of inflammatory bowel diseases (IBD) has greatly evolved, with the more frequent use of immunosuppressants (azathioprine, mercaptopurine, and methotrexate). [START_REF] Lichtenstein | American Gastroenterological Association Institute medical position statement on corticosteroids, immunomodulators, and infliximab in inflammatory bowel disease[END_REF][START_REF] Ardizzone | Randomised controlled trial of azathioprine and 5-aminosalicylic acid for treatment of steroid dependent ulcerative colitis[END_REF][START_REF] Pearson | Azathioprine for maintaining remission of Crohn's disease[END_REF] The thiopurines azathioprine, 6-thioguanine (6-TGN) and mercaptopurine are anti-inflammatory and immunosuppressive drugs that have been available in clinical practice for over half a century. Azathioprine and 6-MP have been shown to be effective for inducing and maintaining clinical remission and fistula closure and for steroid tapering in Crohn's disease (CD). [START_REF] Candy | A controlled double blind study of azathioprine in the management of Crohn's disease[END_REF][START_REF] Markowitz | A multicenter trial of 6mercaptopurine and prednisone in children with newly diagnosed Crohn's disease[END_REF] Thiopurines are now considered standard of care, despite the absence of regulatory approvals. [START_REF] Sandborn | A review of immune modifier therapy for inflammatory bowel disease: azathioprine, 6-mercaptopurine, cyclosporine, and methotrexate[END_REF] Because operative resection of the diseased bowel is not curative, postoperative recurrence remains a problem in patients with CD. In a meta-analysis, Peyrin-Biroulet et al.

found that purine analogs are more effective than placebo in preventing both clinical and endoscopic postoperative recurrence in CD. [START_REF] Peyrin-Biroulet | Azathioprine and 6-mercaptopurine for the prevention of postoperative recurrence in Crohn's disease: a meta-analysis[END_REF] Recently, the ECCO (European Crohn's and Colitis Organisation) consensus stated that in patients with a risk factor for early postoperative recurrence (smoking, prior intestinal surgery, penetrating disease behaviour, perianal location and extensive small bowel resection) the drug of choice is azathioprine/mercaptopurine. [START_REF] Van Assche | The second European evidence-based Consensus on the diagnosis and management of Crohn's disease: Special situations[END_REF] The frequency and patterns of clinical use of thiopurines for the prevention of postoperative recurrence in CD is unknown.

Azathioprine and mercaptopurine are pregnancy Food and Drug Administration (FDA) category D drugs. [START_REF] Van Assche | The second European evidence-based Consensus on the diagnosis and management of Crohn's disease: Special situations[END_REF] Animals studies have demonstrated teratogenicity in mice and rats exposed to azathioprine. [START_REF] Platzek | Dose-response relationship of teratogenicity and prenatal-toxic risk estimation of 6-mercaptopurine riboside in mice[END_REF][START_REF] Mosesso | The genetic toxicology of 6-mercaptopurine[END_REF] In humans, including IBD, multiple case series have not noted an increase in congenital anomalies. Therefore, although thiopurines have FDA rating D, the ECCO consensus recalled that that thiopurines appear to be safe and well tolerated during pregnancy. [START_REF] Van Assche | The second European evidence-based Consensus on the diagnosis and management of Crohn's disease: Special situations[END_REF] However, data on the daily use of azathioprine during pregnancy in women with IBD are scarce. We therefore performed a worldwide survey to evaluate the extent to which gastroenterologists who are experts in the field of IBD are utilizing thiopurines during pregnancy and in the postoperative setting in Crohn's disease. The questionnaire was sent by email to practitioners with expertise in IBD and who have published at least one original article on the use of azathioprine in IBD. In order to locate the email address of the authors, a literature search was conducted using MEDLINE database (U.S. National Library of Medicine, Bethesda, MD, USA) through December 2009, using the following medical subject heading (MeSH) terms: ("Azathioprine"[Title] OR "Mercaptopurine"[Title] OR "Thioguanine"[Title] OR "Thiopurine"[Title]) AND ("Colitis, Ulcerative" OR "Crohn disease" OR "Inflammatory bowel diseases").

MATERIALS AND METHODS

This

Statistical analysis

All quantitative variables are described as medians and percentiles [Interquartile range (IQR), 25-75 th percentile]. All proportions are expressed as percentages with 95% confidence intervals (95% CI). Univariate analysis was performed using the Fischer's exact test. When considering the continuous variables for dichotomous analysis, optimal cut-off values were determined using receiver operating characteristic (ROC) analysis, as described by DeLong et al. [START_REF] Delong | Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach[END_REF] All significant items obtained in univariate analyses were integrated into binary logistic regression model for multivariate analysis using a stepwise method. All variables with Pvalues of < 0.1 were initially included in the model, and variables with P-values of < 0.05 were retained in the model. Results were shown as Odds ratios (ORs) and 95% CI. 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 11.4.1.0 (MedCalc Software, Mariakerke, Belgium).

RESULTS

The literature search found 689 authors who have published at least once on the use of thiopurines in IBD. The questionnaires were first sent to experts on December 20, 2009. In February 2010, 165 questionnaires were received; by April 9, 2010, after the second mailing, we had received a total of 175 questionnaires. Table 1 reports the characteristics of the 175 responding gastroenterologists and their inflammatory bowel disease patients. The median number of IBD patients per physician per year was 400 (IQR 25-75th, 188-600) and the total number of IBD patients followed by all responders was 82,379. A total 21% of questionnaires were sent from the US and 55% from Europe, with a total of 25 different countries worldwide (Figure 1).

In a pregnant woman with a history of severe Crohn's disease in clinical remission after one year on azathioprine, what is your strategy?

In a pregnant woman with a history of severe Crohn's disease in clinical remission since one year on azathioprine, 88.6% (n=155) of experts usually continue azathioprine until delivery, 9.1% (n=16) of physicians never administrate azathioprine during pregnancy, and 2.3% (n=4) of practitioners stop azathioprine treatment only during the third trimester. In multivariate analysis, practitioners who followed more than 100 patients per year were more likely to maintain treatment with azathioprine throughout the pregnancy (OR=8.78; 95% CI, 3.06 to 25.17) while practitioners following less than 100 patients tend to never prescribe azathioprine during pregnancy (OR=13.70; 95% CI, 4.25 to 44.21) (Table 2).

After ileocecal resection for Crohn's disease, what is your strategy regarding azathioprine?

After ileocecal resection for Crohn's disease, 39.4% (n=69) of the responders initiate azathioprine only in patients with high-risk of postoperative recurrence, 28.0% (n=49) prescribe azathioprine according to endoscopic evaluation, 20.0% (n=35) systematically initiate azathioprine, and 12.6% (n=22) had a different strategy. The attitudes of these 22 gastroenterologists regarding the use of azathioprine in the postoperative setting are listed in Table 3.

In multivariate analysis, practitioners who follow less than 200 patients per year tend to prescribe azathioprine according to endoscopic evaluation after ileocecal resection for Crohn's disease (OR=2.56; 95% CI, 1.29 to 5.07). Practitioners were more likely to systematically initiate azathioprine in the postoperative period if the proportion of patients with Crohn's disease in their cohort exceeded 55% (OR=2.73; 95% CI, 1.12 to 6.67), or if they reported having prescribed azathioprine for more than 60% of the patients they follow for Crohn's disease (OR=3.92; 95% CI, 1.73 to 8.90) (Table 2).

DISCUSSION

This is the first survey that evaluated the extent to which gastroenterologists who are experts in the field of IBD are utilizing thiopurines during pregnancy and in the postoperative setting in Crohn's disease. Although thiopurines are considered 'Pregnancy Category D' drugs by the Food and Drug Administration, the ECCO consensus considers azathioprine safe in pregnant women with no consistent reports of abnormalities of fertility, prematurity, or congenital defects. [START_REF] Van Assche | The second European evidence-based Consensus on the diagnosis and management of Crohn's disease: Special situations[END_REF] Consistently, the American Gastroenterological Association Institute recommends, given on the large experience in transplant recipients [START_REF] Polifka | Teratogen update: azathioprine and 6-mercaptopurine[END_REF] and the body of evidence in IBD, the continuation of treatment with azathioprine during pregnancy to keep the mother in remission. [START_REF] Mahadevan | American gastroenterological association institute technical review on the use of gastrointestinal medications in pregnancy[END_REF] A flare of disease during pregnancy may be more deleterious to neonatal outcome than any potential risk from the medication. [START_REF] Mahadevan | American gastroenterological association institute technical review on the use of gastrointestinal medications in pregnancy[END_REF] In a prospective, controlled, multicenter study conducted by the Tel Aviv University (Israel), 189 pregnant women on azathioprine who contacted one of seven teratogen information services were compared to a cohort of 230 pregnant women who contacted two of the seven teratogen information services and took nonteratogenic treatments during their pregnancy. [START_REF] Goldstein | Pregnancy outcome of women exposed to azathioprine during pregnancy[END_REF] The aim of this study was to determine whether exposure to azathioprine during pregnancy increases the risk for major malformations and to determine the effect of azathioprine on pregnancy outcome. [START_REF] Goldstein | Pregnancy outcome of women exposed to azathioprine during pregnancy[END_REF] In this study, although the rate of congenital malformations did not differ between the two groups, more cases of prematurity (21% vs. 5%, P < 0.001) and low birth weight (23% vs. 6%, P < 0.001) were noted in the azathioprine group. [START_REF] Goldstein | Pregnancy outcome of women exposed to azathioprine during pregnancy[END_REF] Data from the Danish nationwide cohort study on women who were exposed to azathioprine or mercaptopurine during pregnancy reported relative risk for induced preterm birth of 4.0 (95% CI 1.5 to 10.8). [START_REF] Langagergaard | Birth outcome in women treated with azathioprine or mercaptopurine during pregnancy: A Danish nationwide cohort study[END_REF] There was no elevated risk of spontaneous preterm birth, low birth weight at term, and congenital abnormalities. [START_REF] Langagergaard | Birth outcome in women treated with azathioprine or mercaptopurine during pregnancy: A Danish nationwide cohort study[END_REF] Moreover, this study suggested that adverse birth outcomes were caused by the underlying disease rather than by use of azathioprine or mercaptopurine. [START_REF] Langagergaard | Birth outcome in women treated with azathioprine or mercaptopurine during pregnancy: A Danish nationwide cohort study[END_REF] Data from the same Danish cohort study on 900 children born to Crohn's disease women between 1996 and 2004, the relative risk of preterm birth and congenital abnormalities in thiopurine exposed women, was 4.2 (95% CI 1.4 to 12.5) and 2.9 (95% CI 0.9 to 8.9), respectively. [START_REF] Norgard | Therapeutic drug use in women with Crohn's disease and birth outcomes: a Danish nationwide cohort study[END_REF] More recently, data from the CESAME study did not show an increased risk of interrupted pregnancies or congenital abnormalities among 204 women who received thiopurines during their pregnancy. [START_REF] Coelho | Pregnancy outcome in patients with inflammatory bowel disease treated with thiopurines: cohort from the CESAME Study[END_REF] In summary, most well conducted studies have shown some excess risk of preterm delivery in pregnant women treated with azathioprine, without warning signals on congenital abnormalities. [START_REF] Goldstein | Pregnancy outcome of women exposed to azathioprine during pregnancy[END_REF][START_REF] Norgard | Therapeutic drug use in women with Crohn's disease and birth outcomes: a Danish nationwide cohort study[END_REF][START_REF] Coelho | Pregnancy outcome in patients with inflammatory bowel disease treated with thiopurines: cohort from the CESAME Study[END_REF] The fact remains that the safety of using azathioprine during pregnancy is a matter of active debate.

In our survey, almost all experts maintain azathioprine during pregnancy in a pregnant woman with a history of severe Crohn's disease in clinical remission after one year on this treatment. The number of patients followed per doctor was predictive of their clinical practice with a clear tendency to maintain thiopurine therapy among practitioners following over 100 patients per year.

Postoperative recurrence of Crohn's disease is a common problem in clinical practice.

After ileocolonic resection, the recurrence rates of Crohn's disease in the neoterminal ileum are 30% and 80% after 3 months and 1 year of evolution, respectively. [START_REF] Ahmed | Pathogenesis of postoperative recurrence in Crohn's disease[END_REF] The rate of clinical recurrence is around 30% with an incremental annual risk of 10%. [START_REF] Ahmed | Pathogenesis of postoperative recurrence in Crohn's disease[END_REF] After a first Crohn's disease-related ileocolonic resection, the risk of second surgery is 15 to 45% at three years, reaching 80% at 15 years. [START_REF] Ahmed | Pathogenesis of postoperative recurrence in Crohn's disease[END_REF] Thiopurines are more effective than mesalamine or imidazole antibiotics alone for preventing both clinical and endoscopic recurrence of Crohn's disease. [START_REF] Van Assche | The second European evidence-based Consensus on the diagnosis and management of Crohn's disease: Special situations[END_REF] According to the ECCO consensus, in patients with a risk factor for early postoperative recurrence, the drug of choice is azathioprine/mercaptopurine. [START_REF] Van Assche | The second European evidence-based Consensus on the diagnosis and management of Crohn's disease: Special situations[END_REF] A meta-analysis of four controlled trials that enrolled 433 patients evaluated the efficacy of purine analogs in the prevention of postoperative recurrence in Crohn's disease. [START_REF] Peyrin-Biroulet | Azathioprine and 6-mercaptopurine for the prevention of postoperative recurrence in Crohn's disease: a meta-analysis[END_REF] In the overall analysis, purine analogs were more effective than control arms in preventing both clinical and severe endoscopic recurrences at 1 year (number needed to treat (NNT)=13 and NNT=7, respectively) and for preventing clinical recurrence at 2 years (NNT=8). [START_REF] Peyrin-Biroulet | Azathioprine and 6-mercaptopurine for the prevention of postoperative recurrence in Crohn's disease: a meta-analysis[END_REF] It seems very likely that due to reasons stated above, two thirds of the experts who responded to this survey advocate the use of azathioprine before any endoscopic evaluation while only one fifth of them initiate a purine analogs therapy according to an endoscopic evaluation. Interestingly, practitioners who follow less than 200 patients per year await the results of endoscopic assessment before introducing azathioprine, while those that follow a cohort composed primarily of patients with Crohn's disease, are more likely to systematically prescribe azathioprine in the postoperative setting.

Nevertheless, in a recent one year, double-blind, randomized study, in patients with postoperative Crohn's disease at high risk of clinical recurrence, superiority for azathioprine versus mesalamine could not be demonstrated for therapeutic failure, defined as a CDAI score > or = 200 and an increase of > or = 60 points from baseline, or study drug discontinuation due to lack of efficacy or intolerable adverse drug reaction. [START_REF] Reinisch | Azathioprine versus mesalazine for prevention of postoperative clinical recurrence in patients with Crohn's disease with endoscopic recurrence: efficacy and safety results of a randomised, double-blind, doubledummy, multicentre trial[END_REF] In this study, clinical recurrence was significantly less frequent with azathioprine in comparison with mesalamine (0/41 (0%) vs. 4/37 (11%), P = 0.031), while study drug discontinuation due to adverse drug reactions only occurred in azathioprine-treated patients (9/41 (22%) vs. 0%, P = 0.002) which had the effect of leveling the therapeutic failure rate between the two arms. [START_REF] Reinisch | Azathioprine versus mesalazine for prevention of postoperative clinical recurrence in patients with Crohn's disease with endoscopic recurrence: efficacy and safety results of a randomised, double-blind, doubledummy, multicentre trial[END_REF] Hence, purine analogs appear to represent an attractive option for patients who can tolerate this drug class. Indeed, in an eight-year intercept cohort of previous or present thiopurine-using IBD patients, a quarter of the patients discontinued thiopurines within 3 months, mostly due to adverse events, however, if thiopurine use was successfully initiated in the first months, its use was usually extended over many years. [START_REF] Jharap | Thiopurine therapy in inflammatory bowel disease patients: analyses of two 8-year intercept cohorts[END_REF] In conclusion, this is the first worldwide IBD expert, statement-based survey that provided information on the extent of use of azathioprine in two clinical situations frequently encountered in clinical practice. During pregnancy, it seems obvious that almost all the experts are unanimous on continued treatment with thiopurines throughout pregnancy. • Country: …………………………………..

• Year of graduation from medical school: ……………….

• Number of IBD patients followed per year: ………………

  to endoscopic evaluation, 20% of gastroenterologists systematically initiate azathioprine, and 12.6% have a different attitude.Conclusions:Almost 9 out of 10 physicians continue azathioprine throughout pregnancy. About 7 out of 10 physicians prescribe azathioprine in the postoperative setting according to ECCO recommendations, whereas one-fifth of practitioners systematically initiate azathioprine after surgery.

11

 11

  was a web-based cross-sectional worldwide survey consisting of multiple-choice and open-ended questions. The questionnaire was developed by the Nancy, Saint-Etienne, and Monash University Hospital Departments of Gastroenterology after an exhaustive review of the literature regarding the use of azathioprine in IBD. The questionnaire consisted of two parts. The first part requested information about the responding practitioner and their IBD patients and the second covered the practitioners' clinical practices (Study questionnaire available as Supplemental document online).

  Page 11 of 19 However, in the postoperative setting, attitudes appear to be less consensual, with two thirds of experts prescribing azathioprine therapy without any postoperative endoscopic evaluation and one third utilizing either an endoscopic-based or an alternative approach.

Figure 1 .

 1 Figure 1. Number of questionnaires received by country

TABLES Table 1 . Characteristics of the 175 responding gastroenterologists and their inflammatory bowel disease patients N Median IQR, 25-75 th

 TABLES1

	Year of graduation from medical school	170	1989	1981 to 1996
	What is the number of IBD patients that you follow per year? 172	400	188 to 600
	Among IBD patients followed per year, what is the proportion of patients with Crohn's disease (%)	163	60	50 to 65
	Among IBD patients you have treated with azathioprine, what is the proportion of patients with Crohn's disease? (%)	171	70	60 to 80
	Among patients that you follow for Crohn's disease, what proportion of those are receiving azathioprine? (%)	173	50	40 to 65
	Among patients that you follow for ulcerative colitis, what proportion of those are receiving azathioprine? (%)	173	30	20 to 40

IBD: inflammatory bowel disease; IQR: interquartile range.

Table 2 . Predictors of gastroenterologists' attitudes according to their characteristics, those of their patients, and access to and reimbursement of thiopurine metabolism monitoring tests (non-significant variables in univariate analysis are not shown) In a pregnant woman with a history of severe Crohn' disease in clinical remission since 1 year on azathioprine, what is your strategy? You stop azathioprine treatment only during the third trimester

 2

	P-value, Univariate a	Cutt-off b	P-value, Multivariate c	Odds ratio	95% CI, Odds ratio

woman with a history of severe Crohn' disease in clinical remission since 1 year on azathioprine, what is your strategy? You never administrate azathioprine during pregnancy P-value, Univariate a Cutt-off b P-value, Multivariate c Odds ratio 95% CI, Odds ratio

	Number of IBD patients followed per year? (n)	0.002	≤100	< 0.0001	13.70	4.25 to 44.21
	In a pregnant

woman with a history of severe Crohn' disease in clinical remission since 1 year on azathioprine, what is your strategy? You usually continue azathioprine until delivery P-value, Univariate a Cutt-off b P-value, Multivariate c Odds ratio 95% CI, Odds ratio

	Number of IBD patients followed per year? (n)	0.003	>100	0.0001	8.78	3.06 to 25.17
	TPMT phenotype (='Access and test reimbursed')	0.03	-	0.04	3.24	1.05 to 10.05

After ileocecal resection for Crohn's disease, what is your strategy regarding azathioprine? You prescribe azathioprine according to endoscopic evaluation P-value, Univariate a Cutt-off b P-value, Multivariate c Odds ratio 95% CI, Odds ratio

	Number of IBD patients followed per year? (n)	0.009	≤200	0.0072	2.56	1.29 to 5.07

After ileocecal resection for Crohn's disease, what is your strategy regarding azathioprine? You systematically initiate azathioprine P-value, Univariate a Cutt-off b P-value, Multivariate c Odds ratio 95% CI, Odds ratio

	F o r
	P
	e
	e r
	Proportion of patients with CD among IBD patients (%) Proportion of patients receiving azathioprine among CD patients R (%) IBD: Inflammatory bowel diseases; CD: Crohn's disease; TPMT: Thiopurine S-methyltransferase. 0.01 >55 0.03 0.03 >60 0.001 e	2.73 3.92	1.12 to 6.67 1.73 to 8.90
	v i e
	w

a Fisher's exact test

•

  Among patients you have treated with azathioprine, what are the respective proportions of ulcerative colitis and Crohn's disease? (the sum must equal 100%): Among patients that you follow for UC, what proportion of those are receiving azathioprine? _____% • Among patients that you follow for CD, what proportion of those are receiving azathioprine? QUESTION N°2 In a pregnant woman with a history of severe Crohn' disease in clinical remission since 1 year on azathioprine, what is your strategy: • You never administrate azathioprine during pregnancy yes no • You stop azathioprine treatment only during the third trimester yes no • You usually continue azathioprine until delivery yes no Question N°3 After ileocecal resection for Crohn's disease, what is your strategy regarding azathioprine: • You systematically initiate azathioprine yes no • You initiate azathioprine only in patients with high-risk of postoperative recurrence yes no • You prescribe azathioprine according to endoscopic evaluation yes no • You have a different attitude (Thank you for giving the details on your attitude): ……………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………… THANK YOU!

	-Ulcerative colitis =	________%
	-Crohn's disease = F o ________% r • _____% P
	e Do you have access to the following azathioprine pharmacogenomic tests? QUESTION N°1 TPMT genotype e r
	• Access and test reimbursed • Access but test not reimbursed	R
	• No access		e
	TPMT phenotype • Access and test reimbursed • Access but test not reimbursed • No access 6-TGN dosage	v i e w
	• Access and test reimbursed
	• Access but test not reimbursed
	• No access
	Methylated derivatives dosage
	• Access and test reimbursed
	• Access but test not reimbursed
	• No access

ACKNOWLEDGEMENTS

We are grateful to all our colleagues who participated in the survey.

Authors' declaration of personal interests: LPB, AO, XR, MS declare no conflict of interest.

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

b

Cut-off defined using the receiver operating characteristic analysis as described by DeLong et al. 12 c Binary logistic regression


Jacopo Grazzini

Antonio Turiel

Hussein Yahia

Isabelle Herlin
email: isabelle.herlin@inria.frturiel@icm.csic.es

† Clime

EDGE-PRESERVING SMOOTHING OF HIGH-RESOLUTION IMAGES WITH A PARTIAL MULTIFRACTAL RECONSTRUCTION SCHEME

Keywords: High-resolution, Edge-preserving, Smoothing, Pre-segmentation, Multiscale, Multifractal, Singularity, Reconstruction

The new generation of satellites leads to the arrival of very high-resolution images which offer a new quality of detailed information about the Earth's surface. However, the exploitation of such data becomes more complicated and less efficient as a consequence of the great heterogeneity of the objects displayed. In this paper, we address the problem of edgepreserving smoothing of high-resolution satellite images. We introduce a novel approach as a preprocessing step for feature extraction and/or image segmentation. The method is derived from the multifractal formalism proposed for image compression. This process consists in smoothing heterogeneous areas while preserving the main edges of the image. It is performed in 2 steps: 1) a multifractal decomposition scheme allows to extract the most informative subset of the image, which consists essentially in the edges of the objects; 2) a propagation scheme performed over this subset allows to reconstruct an approximation of the original image with a more uniform distribution of luminance. The strategy we adopt is described in the following and some results are presented for Spot acquisitions with spatial resolution of 20 × 20 m 2 .

INTRODUCTION

In the latest years, the improvement of the technology for observing the Earth from the space has led to a new class of images with very high spatial resolution. New satellite sensors are getting closer to the spatial requirements, with, for example, resolutions of 2.5 m (Spot), 1 m (Ikonos) or even 0.7 m (QuickBird). High resolution (HR) imagery offers a new quality of detailed information about the properties of the Earth's surface together with their geographical relationships. Consequently, this has given rise to a growing interest on image processing tools and their application on this kind of images [START_REF] Mather | Computer Processing of Remotely-Sensed Images: An Introduction[END_REF]. Smaller and smaller objects (such as house plots, streets...), as well as precise contours of larger objects (such as field structures) are now available, automatic methods for extracting these objects are of great interest. However, due to the fact that HR images show great heterogeneity, standard techniques for analyzing, segmenting and classifying the data become less and less efficient. When the resolution increases, the spectral within-field variability also increases, which can affect the accuracy of further classification or segmentation schemes [START_REF] Schiewe | Segmentation of high-resolution remotely sensed data -concepts, applications and problems[END_REF]: images contain more complicated and detailed local textures; characteristic objects of the images (fields, buildings, rivers) are no more homogeneous. Thus, classical approaches cannot produce satisfactory results because they may induce simultaneously under-and over-segmentation within a single scene, depending on the heterogeneity of the considered objects, that confuse the global information and prevent further analysis. Generally several preprocessing steps may be required before such methods can be applied [START_REF] Schowengerdt | Remote Sensing. Models and Methods for Image Processing[END_REF].

This paper introduces a new approach to edge-preserving smoothing of HR images as a preprocessing step for feature extraction and/or image segmentation. The problem can be related with the idea of resolution reduction: the retained technique should enable to preserve the features of the original image corresponding to the boundaries of the objects while homogeneizing the other parts (non-edges) of the image. Recently, Laporterie-Déjean et al. [START_REF] Laporterie-Déjean | Pre-segmentation of high-resolution images thanks to the morphological pyramid[END_REF] proposed a multiscale method for presegmenting HR images. The algorithm consists in performing a non-exact reconstruction: it decomposes and partially reconstructs images using morphological pyramids that enable to extract details wich regard their structures within the image.

Like in [START_REF] Laporterie-Déjean | Pre-segmentation of high-resolution images thanks to the morphological pyramid[END_REF], we address this problem with a method derived from data compression. We use the multifractal approach introduced in [START_REF] Turiel | The multi-fractal structure of contrast changes in natural images: from sharp edges to textures[END_REF] as a resolution-reducing device. Like many image processing techniques, it makes use of the image edge information that is contained in the image gradient. Such an approach is ideal, as it assumes that objects can be reconstructed from their boundary information [START_REF] Turiel | Reconstructing images from their most singular fractal set[END_REF]. Moreover, it constitutes a multiscale approach which is well recognized to attain the best performance in processing purposes over satellite data, like image fusion and image merging [START_REF] Yocki | Image merging and data fusion by means of the discrete 2D wavelet transform[END_REF]. Even on a single scene, different scales of analysis are needed, depending on the homogenity of the objects under consideration and on the desired final application. The process we finally propose is done in two steps:

• First, meaningful subsets of the original image, which mainly consists in its boundaries, are extracted using a multifractal decomposition scheme. In this scene, we can recognize some fields, roads and also poorly delimited areas which correspond to cities.

• Second, an universal propagation kernel is used to partially reconstruct typical object shapes from the values of the gradient of the original image over the previous subset.

The paper is organized as follows. In section 2, we review the multifractal approach for extracting the meaningful entities in the images. In section 3, we introduce the method for reconstructing the images. We show that this method provides good results on Spot images with spatial resolution of 20 × 20 m 2 (see Fig. 1) and that it could be applied to any kind of data. We also consider an alternative version of this method. As a conclusion, we will discuss the advantages of our methodology.

THE MULTIFRACTAL DECOMPOSITION

Multifractality is a property of turbulent-like systems which is generally reported on intensive, scalar variables of chaotic structure [START_REF] Frisch | Turbulence. The Legacy of Kolmogorov[END_REF]. However, methods based on this approach have also shown to produce meaningful results in the context of real world images where irregular graylevel transitions are analyzed [START_REF] Turiel | The multi-fractal structure of contrast changes in natural images: from sharp edges to textures[END_REF]. Natural images can be characterized by their singularities, i.e. the set of pixels over which the most drastic changes in graylevel value occur. In [START_REF] Turiel | The multi-fractal structure of contrast changes in natural images: from sharp edges to textures[END_REF], Turiel and Parga introduced a novel technique based on multifractal formalism to analyze these singularities. We develop in the following the basis of this technique.

In this approach, the points in a given image are hierarchically classified according to the strength of the transtion of the image around them. The first step concerns the definition of an appropriate multifractal measure. Let us denote I an image and ∇I its spatial derivative. We then define a measure µ through its density dµ(x) = dx |∇I|(x), so that the measure of a ball B r (x) of radius r centered around the point x is given by:

µ(B r (x)) = Br(x)
dy |∇I|(y).

(1)

Here |∇I| denotes the modulus of the gradient ∇I. This measure gives an idea of the local variability of the graylevel values around the point x. It was observed that for a large class of natural images, such a measure is multifractal [START_REF] Turiel | The multi-fractal structure of contrast changes in natural images: from sharp edges to textures[END_REF], i.e. the following relation holds: x) .

µ(B r (x)) ∼ α(x) r 2+h(
(2)

All the scale dependance in eq. ( 2) is provided by the term r 2+h(x) where the exponent h(x) depends on the particular point considered. This exponent is called singularity exponent and quantifies the multifractal behaviour of the mesure µ. The second step of the approach regards the estimation of these local exponents. This is done through a wavelet projection µ (Daubechies, 1992) of the measure, for which the same kind of relation as eq. ( 2) holds in a continuous framework, so that the exponents h(x) can be easily retrieved. The reader is refered to [START_REF] Turiel | The multi-fractal structure of contrast changes in natural images: from sharp edges to textures[END_REF] for a full description of the method. By this way, each pixel x of the image can be assigned a feature which measures the local degree of regularity of the signal ∇I at this point. The singularity exponents computed on the Spot image of the Fig. 1 are represented in the Fig. 2. We see that the lowest values of singularity are mainly concentrated near the boundaries of the objects of the image (large culture fields in the middle part, roads in the lower left part). Some of them are also visible inside the culture fields, as they are not completely flatly illuminated, and in city areas (upper right part) that are rather heterogeneous. The image can then be hierarchically decomposed in different subsets F h gathering the pixels of similar features:

F h ≡ {x | h(x) ≈ h} . (3)
We know, from the multifractal theory, that each one of these subsets is fractal, i.e. it exhibits the same geometrical structure at different scales. In particular, the arising decomposition allows isolating a meaningful fractal component, the so-called MSM (Most Singular Component, denoted F ∞ ) associated with the strongest singularities (i.e. the lowest -or most negative -exponent, denoted h ∞ ). In Fig. 3, the MSM was computed on the original image at a rather coarse resolution: the pixels for which h ∞ = -0.42 ± 0.3 were included in the MSM. From information theory, the MSM can be interpreted as the most relevant, informative set in the image [START_REF] Turiel | Reconstructing images from their most singular fractal set[END_REF]. In [START_REF] Grazzini | Entropy estimation and multiscale processing in meteorological satellite images[END_REF], Grazzini et al. have evidencied that this subset, computed on meteorological satellite data, is related with the maxima of some well-defined local entropy. From multifractal theory, the MSM can be regarded as a reconstructing manifold. In [START_REF] Turiel | Reconstructing images from their most singular fractal set[END_REF], Turiel and del Pozo have shown that using only the information contained by the MSM, it is possible to predict the value of the intensity field at every point.

We describe the algorithm for reconstructing images in the next section and we show that it can be used as a technique for edge-preserving smoothing of images.

THE RECONSTRUCTED IMAGES

In [START_REF] Turiel | Reconstructing images from their most singular fractal set[END_REF], Turiel and del Pozo proposed an algorithm supposed to produce a perfect reconstruction FRI (Fully Reconstructed Image) from the MSM. The authors introduced a rather simple vectorial kernel g capable to reconstruct the signal I from the value of its spatial gradient ∇I over the MSM. Namely, let us define the density function δ ∞ (x) of the subset F ∞ which equals

1 if x ∈ F ∞ and 0 if x / ∈ F ∞ .
Let also define the gradient restricted to the same set v ∞ ≡ ∇I δ ∞ , i.e. the field which equals the gradient of the original image over the MSM and is null elsewhere: this will be the only data required for the reconstruction. The reconstruction formula is then expressed as:

I(x) = g v ∞ (x) (4)
where denotes the convolution. The reconstruction kernel g is easily represented in Fourier space by:

ˆ g(f ) = i f / |f | 2 (5)
where ˆstands for the Fourier transform, f denotes the frequency and i the imaginary unit. The principle is that of a propagation of the values of the signal ∇I over the MSM to the whole image. The full algorithm for reconstruction is given in [START_REF] Turiel | Reconstructing images from their most singular fractal set[END_REF]).

The multifractal model described above also allows to consider a related concept, that of RMI (Reduced Multifractal Image), as it was introduced in [START_REF] Grazzini | Entropy estimation and multiscale processing in meteorological satellite images[END_REF]. It consists in propagating, through the same reconstruction formula (4), instead of v ∞ , another field v 0 so that a more uniform distribution of the luminance in the image is obtained. Namely, the field v 0 is simply built by assigning to every point in the MSM an unitary vector, perpendicular to the MSM and with the same orientation as the original gradient ∇I. Thus, by introducing in eq. ( 4) this rather naive vector field, we obtain the RMI. Consequently, the RMI has the same multifractal exponents (and so, the same MSM) as the original image, but, as desired, a more uniform graylevel distribution. In fact, this image ideally attains the most uniform distribution of graylevels compatible with the multifractal structure of the original image. RMI images define smaller (i.e., more compressed) codes than FRI images; both images are very similar when the original images are uniformly illuminated.

In Fig. 3, we present the images reconstructed from the fields v ∞ and v 0 defined by the MSM previously computed (see section 2). This approximates the original image of Fig. 1 with a Peak Signal to Noise Ratio (P SN R) of 24.93 dB and P SN R = 21.34 dB respectively.

DISCUSSION

From the whole reconstructions displayed in Fig. 3 and the details displayed in Fig. 4, we see that:

• for both reconstructions, there is a high degree of smoothing in rather homogeneous areas,

• main edges are preserved, even those ones being represented by small gray value changes, like inside the culture fields,

• even if it may happen that edges between small areas are deleted, like for small culture fields, small homogeneous regions are generally conserved.

The similarity between both reconstructions is mainly due to the fact that on this kind of images (namely, land cover images with rather linear edges induced by culture fields) the field v 0 does not strongly deviate from the field v ∞ . So, in first approximation, the RMI could be used for providing the information about the boundaries. However, the corresponding graylevel distributions (Fig. 4) show that the FRI provides a good smoothed version of the original image, with suppression of peaks in the distribution, whereas the RMI provides a completely different chromatic distribution. The RMI represents a different, possible view of the same scene, with the same objects and the same geometry as the FRI, but different distributed illumination of each part. Namely, the differences between the RMI and the original image are only due to these differences in illumination. In spite of the advantages of a more compact code as the one associated to the RMI, we finally retain the FRI as the convenient edge-preserving smoothing approximation of the original image: it cleans up noise in the homogeneous areas but preserves important structures and also preserves the luminance distribution. Besides, the quality of the approximation of the original image is good for the FRI, which is an essential requirement for further processing like feature extraction. Close inspection to the image also shows that the method is able to enhance subtle texture regions, like small culture fields. It is clear that the results are superior to the results of a simple method like pixel averaging for instance. Now, one of the major advantages of our method of presegmentation is that it is parameterizable. We should notice that the reconstruction algorithm for computing the FRI defined by the eq. ( 4) is linear [START_REF] Turiel | Reconstructing images from their most singular fractal set[END_REF]. It means that, if the reconstructing manifold is the union of two subsets ℘ 1 ∪ ℘ 2 , then the FRI ℘1∪℘2 reconstructed from this manifold equals the addition of the FRI ℘1 and FRI ℘2 reconstructed from each part separately. Thus, the more singular pixels the reconstructing manifold gathers, the closer to the original image the FRI is. In particular, when the MSM consists of all the points in the image, eq. ( 4) becomes a trivial identity and the reconstruction is perfect. So, we can approximate the original image as close as desired. We just need to choose the density of the manifold used for the reconstruction, i.e. we need to adjust the range of values accepted for the singularity exponents of the pixels belonging to the MSM. By this way, the degree of smoothing of objects in the image can be controlled. On the Fig. 5, we see that the details of the image are incorporated in the reconstruction when increasing the authorized number of pixels in the MSM, and this is done gradually, according to their degree of singularity.

CONCLUSION

In this paper, we have proposed to perform a pre-segmentation of high resolution images prior to any processing. For this purpose, we adopt an approach related with data compression and based on the multifractal analysis of images.

The main idea is that of a partial reconstruction process of the images from the extraction of their most important features.

The multifractal algorithm is performed in two steps, which consist in: first, extracting the most singular subset of the image, i.e. the set of pixels where strong transitions of the original image occur, and, then, performing a reconstruction by propagating the graylevel values of the spatial gradient of the image from this subset to the other parts. The multiscale character of the extraction step allows to retain the relevant edges, no matter at which scale they happen, and without significant artifacts. The most singular subset is mainly composed of pixels belonging to the boundaries of the objects in the image. So that, our algorithm to reconstruct images is consistent with classical hypothesis stating that edges are the most informative parts of the image [START_REF] Marr | Figure 5: FRI obtained considering different MSM with varying density in the image[END_REF]. The quality of the reconstruction depends on the validity of the hypothesis defining the reconstruction kernel and on the accuracy of the edge detection step. It should be also noticed that this method can be used as a starting point for a coding scheme, as it retains the most meaningful features of the image.

The reconstruction strategy results in very nicely smoothed homogeneous areas while it preserves the main information contained in the boundaries of objects. It is good at enhancing textures, as it smoothens the image, and, thus, suppresses small elements corresponding to the main heterogeneity. The image structures are not geometrically damaged, what might be fatal for further processings like classification or segmentation. Indeed, it creates homogeneous regions instead of points or pixels as carriers of features which should be introduced in further processing stages. Moreover, the reconstruction is parameterizable. The fidelity to the original image can be adjusted, by gradually incorporating as many details of the original image as desired, so that the degree of smoothing can be controlled. For a deeper validation of the method, we should perform the algorithm on very high resolution images.

Figure 1 :

 1 Figure 1: High-resolution Spot image (20×20 m per pixel) acquired in the Near Infrared channel. In this scene, we can recognize some fields, roads and also poorly delimited areas which correspond to cities.

Figure 2 :

 2 Figure 2: Left: representation of the singularity exponents computed on the image of the Fig. 1 in the range [0, 255] of graylevel values; the brighter the pixel, the greater the singularity exponent a this point. Right: corresponding distribution of the singularity exponent; the range of values obtained is [-0.80, 0.81].

Figure 3 :

 3 Figure 3: Top: MSM (dark pixels) extracted at h ∞ = -0.42 ± 0.3 from the image of Fig. 1; quantitatively, the MSM gathers 22.29% of the pixels of the image. Middle: FRI computed with eq. (4) on the field v ∞ (P SN R = 24.93 dB). Bottom: RMI computed with the field v 0 (P SN R = 21.34 dB).

Figure 4 :

 4 Figure 4: Details of the reconstructed images and their graylevel values' distribution. From top to bottom: details of the original Spot image, details of the FRI and the RMI, respectively reconstructed with the fields v ∞ and v 0 .