Solesne Papillard-Marechal

Vincent Enouf

Aurélie Schnuriger

Astrid Vabret

Edouard Macheras

Marie-Anne Rameix-Welti

Bernard Page

Elyanne ; Gault

Monitoring epidemic viral

Keywords: orthomyxovirus, paramyxovirus, molecular diagnosis Orthomyxovirus, Paramyxovirus, Molecular diagnosis 58 59

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Monitoring epidemic viral respiratory infections using one-step real-time triplex RT-1 PCR targeting influenza A and B viruses and respiratory syncytial virus 2

Running title: Real-time triplex PCR for influenza A/B and RSV 3 4 Solesne Papillard-Marechal 1 , Vincent Enouf 2,3,4,5,* , Aurélie Schnuriger 6,7* , Astrid Vabret 8 , 5

Edouard Macheras 9,10 , Marie-Anne Rameix-Welti 9,10 , Bernard Page 11 , François Freymuth 8 , 6

Sylvie van der Werf 2,3,4,5 , Antoine Garbarg-Chenon 6,7 , Bertrand Chevallier 1 , Jean-Louis 7 Gaillard 9,10 , Elyanne Gault The seasonal epidemic circulation of influenza A, influenza B and human respiratory 61 syncytial virus (RSV) viruses causes a large part of the burden of viral respiratory infections 62 in the northern hemisphere, especially that involving infants under 96 weeks of age and 63 elderly patients [Nair et al., 2010;[START_REF] Neuzil | The effect of 327 influenza on hospitalizations, outpatient visits, and courses of antibiotics in children[END_REF][START_REF] Poehling | The underrecognized burden of influenza in young children[END_REF][START_REF] Simoes | Respiratory syncytial virus infection[END_REF]64 Simonsen et al., 2000]. Rapid identification of the viral cause of acute respiratory infection 65 has consequences for patient monitoring and care, including hospital admission or discharge, 66 administration of antibiotics and antiviral therapy, and reduction of diagnosis investigation 67 [START_REF] Adcock | Effect of rapid viral diagnosis on the 272 management of children hospitalized with lower respiratory tract infection[END_REF][START_REF] Barenfanger | Clinical and financial benefits of 275 rapid detection of respiratory viruses: an outcomes study[END_REF]. Antigen detection by immunofluorescence 68 (IF) or immunochromatography is the usual first-line diagnostic approach, although it lacks 69 sensitivity [START_REF] Louie | Rapid influenza antigen test for diagnosis of pandemic (H1N1) 319 2009[END_REF][START_REF] Steininger | Effectiveness of 337 reverse transcription-PCR, virus isolation, and enzyme-linked immunosorbent assay 338 for diagnosis of influenza A virus infection in different age groups[END_REF]]. An alternative is to test for viral 70 genomes by molecular means. Although various in-house and commercial assays are 71 available, first-line molecular detection is rarely routinely practiced, either because of high 72 cost (commercial assays) or because few assays are multiplex and have short turnaround 73 times [START_REF] Bellau-Pujol | Development of three multiplex RT-PCR assays for 279 the detection of 12 respiratory RNA viruses[END_REF][START_REF] Bonroy | Use of a multiplex real-time 281 PCR to study the incidence of human metapneumovirus and human respiratory 282 syncytial virus infections during two winter seasons in a Belgian paediatric hospital[END_REF][START_REF] Brittain-Long | Multiplex 291 real-time PCR for detection of respiratory tract infections[END_REF]Gunson et al., 74 2005;[START_REF] Kuypers | 311 Comparison of real-time PCR assays with fluorescent-antibody assays for diagnosis of 312 respiratory virus infections in children[END_REF][START_REF] Van De Pol | Increased detection of respiratory syncytial virus, influenza viruses, 344 parainfluenza viruses, and adenoviruses with real-time PCR in samples from patients 345 with respiratory symptoms[END_REF]. 75

In this study, a simple, sensitive and specific one-step triplex real-time RT-PCR assay was 76 developed to detect the genomes of RSV, influenza A (including the pandemic influenza 77 A(H1N1) 2009 virus) and influenza B simultaneously in respiratory samples. This assay was 78 performed daily during the 2008-2009 and 2009-2010

MATERIALS AND METHODS 83

Viruses 84

Various influenza A, influenza B and RSVA/B strains were used to determine sensitivity and 85 efficiency of the assay (table 1). RSV-A of genotypes 1 to 5 and RSV-B of genotypes 1 to 6, 86 isolated from clinical specimens and collected between 1986 and 2003 at the Hôpital 87 Clémenceau, Caen, France were tested. RSV genotypes were determined by phylogenetic 88 analysis of the G gene, according to the protocol established by the National Paramyxoviridae 89 Centre (AV and FF, unpublished data) using primers described previously [Zambon et al., 90 2001]. Viruses were propagated in cell culture (MDCK for influenza A and influenza B, 91 MRC5 for RSV) and virus titres are reported as plaque forming units (pfu) (influenza A and 92 B) or the 50% tissue culture infectious dose (TCID 50 ) /mL (RSV). 93

Undiluted culture supernatants of PIV-1, -2, -3 and -4, hMPV-B, adenovirus type-1, echovirus 94 type-30, rhinovirus type-31, and coronavirus OC43 and 229E were used to assess the 95 specificity of the assay. 96

Clinical samples 97

Specimens (nasopharyngeal aspirates or swabs) were collected from patients with acute 98 respiratory infection or influenza-like syndrome and tested daily either at Hôpital Ambroise 99 Paré, a general teaching hospital, or at Hôpital Trousseau, a paediatric teaching hospital. 100 The cycling conditions for real-time RT-PCR were those recommended by Argene. 127

One-step real-time triplex RT-PCR for influenza

Sub-typing of influenza A viruses: 128

Samples were used to inoculate MDCK. Influenza viruses were detected by 129 haemagglutination assay with guinea-pig erythrocytes. Isolate subtype was determined by 130 haemagglutination-inhibition, as described previously [START_REF] Kendal | Laboratory-based surveillance of 306 influenza virus in the United States during the winter of 1977-1978. I. Periods of 307 prevalence of H1N1 and H3N2 influenza A strains, their relative rates of isolation in 308 different age groups, and detection of antigenic variants[END_REF]. 131 3). The temporal distribution of virus detection is shown in figure 171 1. Of the 298 patients tested, 188 (63%) were positive: 145 (78%) for RSV, 42 (22%) for 172 influenza A and one for both RSV and influenza A. No influenza B infection was detected 173 during this period. For influenza A, cell-culture isolation and subsequent sub-typing was 174 performed for 23 (56%) samples: all corresponded to the epidemic strain current at that time: 175 A/Brisbane/10/2007 (H3N2). Positive cell-culture isolation was significantly correlated with 176 viral load in the specimen (viral load being estimated from C T values). In cell-culture positive 177 specimens, the median C T value was 27.09, whereas in cell-culture negative specimens it was 178 32.57 (P<0.0001). 179

The period between week 2009-36 and week 2010-04 was characterized by the pandemic 180 circulation of influenza A(H1N1) 2009. From week 2009-43 to 2010-04, the test was also 181 used in the Hôpital Trousseau laboratory (table 3). Of 776 patients (adults and children) tested 182 at Hôpital Ambroise Paré, 316 (41%) were positive: 183 (58%) for RSV, 132 (22%) for 183 influenza A and one for both RSV and influenza A (figure 2A). Of 1,343 patients (only 184 children) tested at Hôpital Trousseau, 663 (49%) were positive: 501 (76%) for RSV, 151 185 (23%) for influenza A, and eleven for mixed RSV-influenza A infections (figure 2B). Of the 186 The detection of RSV infection in infants under six weeks old, or presenting with a medical 191 condition such as prematurity or underlying cardiopulmonary disease, leads to hospital 192 admission to monitor the risk of rapid respiratory aggravation or apnoea [START_REF] Hall | Nosocomial respiratory syncytial virus infections: the "Cold War" has not 301 ended[END_REF]193 Tregoning and Schwarze, 2010]. Also, children with influenza often present with alarming 194 neurological symptoms associated with respiratory disease, but will not need further 195 neurological investigation if influenza virus is detected rapidly [2009b]. Similarly, if a viral 196 infection is diagnosed in a newborn with isolated fever, investigations for neonatal bacterial 197 infection can be limited. Moreover, early administration of neuraminidase inhibitors in cases 198 of influenza may prevent severe forms of infection, as reported during the influenza A( H1N1 ) 199 2009 pandemic [2009a; 2009c]. Finally, when RSV and influenza viruses co-circulate at an 200 epidemic rate (as was the case in 2008-2009 and 2009-2010), it may not be possible to 201 distinguish infection by one virus from that by the other on the basis of clinical presentation 202 [START_REF] Bosis | Role of respiratory pathogens in infants hospitalized 286 for a first episode of wheezing and their impact on recurrences[END_REF][START_REF] Bourgeois | Relative impact of influenza and 289 respiratory syncytial virus in young children[END_REF]. Thus, rapid and sensitive tools are needed 203 specifically to detect influenza and RSV infections, and to identify mixed infections. The 204 diagnostic value of rapid antigen detection tests for influenza and RSV infections remains a 205 subject of debate: although technically well adapted to the management of acute respiratory 206 infections, the currently available tests lack sensitivity and specificity [START_REF] Louie | Rapid influenza antigen test for diagnosis of pandemic (H1N1) 319 2009[END_REF]207 Steininger et al., 2002]. 208

The aim of this study was to improve the efficiency of diagnosis of acute respiratory 209 infections, in a context of high rates of infection by three major pathogens, which can be 210 involved in mixed infections. The higher sensitivity of RT-PCR compared to antigen 211 detection prompted us to use this technology to develop an inexpensive (<10.00 € / test), rapid 212 (<4h), specific and sensitive assay, targeting the genomes of influenza A, influenza B, and 213 RSV. Triplex RT-PCR is cheaper and faster than three simplex assays. To facilitate use in 214 Pure Compact system (Roche Diagnostics, Meylan, France), and the assay was successfully 216 transferred to a different laboratory setting, where a different real-time PCR thermocycler was 217 used (ABI PRISM® 7500 Real Time PCR System -Applera). Although not to be compared 218 to rapid antigen testing, the assay was efficient enough to provide clinicians with specific and 219 sensitive results daily: for samples made available at 13:00, the results were returned by 220 16:30. 221 The assay was designed to detect simultaneously the presence of the three viral genomes, and 222 could thus identify mixed infections. Its ability to detect various strains of viruses was 223 assessed with a panel of viruses isolated in cell-culture. With this panel, the sensitivity and 224 efficiency of the triplex RT-PCR were found to be slightly lower than those obtained with the 225 equivalent simplex assays, although the difference was not significant (results not shown), as 226 previously reported by others for multiplex RT-PCR [START_REF] Bonroy | Use of a multiplex real-time 281 PCR to study the incidence of human metapneumovirus and human respiratory 282 syncytial virus infections during two winter seasons in a Belgian paediatric hospital[END_REF]. However, the 227 sensitivity and efficiency of the assay were suitable for diagnosis. Indeed, the results were 228 concordant for 100% of the 75 samples found positive with commercial RT-PCR assays, and 229 the triplex assay successfully detected mixed infections. The 7900HT thermocycler allows no 230 more than three different fluorescent dyes in real-time PCR assays, so the triplex assay could 231 not also include an internal control. However, no PCR inhibitors were detected in 132 232 samples tested by the Argene method, indicating that the risk of missing a positive sample 233 was small. If necessary for routine diagnosis, an internal control could be added to the sample 234 prior to extraction, and the corresponding control RT-PCR could then be performed in 235 parallel during the same run. The results obtained with this assay in daily practice during the 236 2008-2009 and 2009-2010 seasons were consistent with the data provided by the national 237 influenza-monitoring network of GPs and paediatricians GROG (Groupes Régionaux 238 d'Observation de la Grippe -Regional Influenza Surveillance Grouphttp://www.grog.org), 239 and by the National Influenza Centre (Northern France), indicating, in particular, that 240 circulation of influenza B was sporadic or absent in France during the periods studied. As 241 previously described [START_REF] Bourgeois | Relative impact of influenza and 289 respiratory syncytial virus in young children[END_REF], RSV infection was more frequent than 242 influenza in young children, as evidenced by the higher percentage of RSV-positive 243 specimens, even during the influenza A(H1N1) pandemics. Finally, the availability of a 244 molecular tool allowing to distinguish rapidly influenza A from RSV in two general hospital 245 settings in the context of the influenza A(H1N1) pandemics was of utmost value. 246

In conclusion, the user-friendly in-house triplex RT-PCR reported here was efficient for 247 testing nasopharyngeal specimens for current epidemic strains of influenza A (including 248 seasonal H1N1, predominant in 2007H1N1, predominant in -2008H1N1, predominant in [Vaux, 2008]], seasonal H3N2, as sub-typed by 249 cell-culture during the 2008-2009 season and pandemic influenza A(H1N1) 2009), influenza 250 B, RSV-A and RSV-B. Its ability to detect mixed infections also facilitates investigations to 251 increase our knowledge about the incidence and severity of influenza A and RSV co-252 infections [START_REF] Coiras | Simultaneous detection of 293 fourteen respiratory viruses in clinical specimens by two multiplex reverse 294 transcription nested-PCR assays[END_REF][START_REF] Kehl | Evaluation of the Hexaplex assay for detection 303 of respiratory viruses in children[END_REF] a RT-PCR efficiency was calculated for each strain, as previously described, by testing tenfold serial dilutions of the corresponding RNAs [START_REF] Gault | 296 Quantification of human cytomegalovirus DNA by real-time PCR[END_REF][START_REF] Gal | Quantification 314 of hepatitis delta virus RNA in serum by consensus real-time PCR indicates different 315 patterns of virological response to interferon therapy in chronically infected patients[END_REF].

b Ga1 to Ga5 are RSV-A strains of genotypes 1 to 5.

c Gb1 to Gb6 are RSV-B strains of genotypes 1 to 6.

  Monitoring epidemic viral respiratory infections using onestep real-time triplex RT-PCR targeting influenza A and B viruses and respiratory syncytial virus triplex PCR for influenza A/B and RSV Papillard-Marechal et al 1

  A, influenza B and RSVA/B genome 101 detection in respiratory samples 102 RNA was extracted with the QIAamp Viral-RNA Mini-Kit (Qiagen, Courtaboeuf, France) 103 from 140µL of fresh or thawed specimen. Aliquots of the resulting RNA preparation (7.5µL 104 of 60µL eluted) were included in 25µL of RT-PCR mixture containing primers and probes 105 (table 2), and the TaqMan® One-Step RT-PCR Master Mix Reagents Kit (Applera, 106 Courtaboeuf, France). Each run included a negative control and a triplex positive control; the 107 triplex PCR for influenza A/B and RSV Papillard-Marechal et al positive controls contained 2.5µL of each target RNA extracted from culture supernatants and 108 diluted to give a positive signal of 32 C T s, corresponding to an input of approximately 1,000 109 pfu for influenza A and B, and 20 TCID50 for RSV (table 1). The reaction consisted of 30 110 min of reverse-transcription at 48°C, 10 min of activation at 95°C, and 45 cycles of 111 amplification (95°C for 15 s and 59°C for 90 s). A 7900HT Fast Real-Time PCR System 112 (Applera) was used for real-time RT-PCR. Reaction giving C T values ≤44 were considered to 113 be positive. 114 Other virological methods 115 IF antigen detection: IF tests were performed on freshly sampled nasopharyngeal aspirates. 116 Anti-influenza A, -influenza B and -RSV monoclonal antibodies (Argene S.A., Verniolle, 117 France) were used as recommended by the manufacturer. 118 Commercial real-time RT-PCR assays: primers and probes of the Flu-A/B r-gene™ and RSV-119 A/B r-gene™ (Argene S.A.) assays were used in two independent RT-PCR targeting either 120 influenzaA/B or RSV-A/RSV-B. Aliquots of RNA preparations (10µL, see procedure above) 121 were added to two RT-PCR mixtures containing 14µL of primers and probes and 1µL of 122 OneStep RT-PCR mix (Qiagen). Internal control IC2 (DICO Extra r-gene™, Argene S.A.) 123 was added to the sample prior to extraction, to monitor the extraction step and to detect PCR 124 inhibitors. An additional RT-PCR mixture containing 10µL of RNA extract, 15µL of the 125 DICO Extra r-gene premix and 0.17µL of the OneStep RT-PCR mix (Qiagen) was prepared. 126

  triplex PCR for influenza A/B and RSV Papillard-Marechal et al 7 RESULTS 141 Specificity, sensitivity and efficiency of the one-step real-time triplex RT-PCR assay 142 The specificity of the triplex assay was confirmed by the negative results obtained with viral 143 genomes of ten other respiratory viruses. The triplex positive control gave repeatedly positive 144 signals for the three viruses targeted. No cross-amplification was observed in the triplex assay 145 when tested with individual control influenza A, influenza B and RSV genomes. The 146 sensitivity of the assay varied according to the strain, from 1 to 10 pfu/input for influenza 147 viruses and was 0.02 TCID 50 /input for RSV (table 1). 148 The ability of the one-step real-time triplex RT-PCR assay to detect viral genomes in 149 nasopharyngeal samples was assessed on a selected panel of 182 samples that were collected 150 and tested by IF in 2007-2008 and stored at -80°C. These samples were tested both by the in-151 house triplex RT-PCR assay and the commercial Flu A/B and RSV A/B r-gene™ assays. The 152 DICO Extra r-gene™ control was used to detect PCR inhibitors. All samples positive by IF 153 (n=37) for influenza A (n= 8), influenza B (n= 3) or RSV (n= 26) were found positive for the 154 respective viruses with both molecular tests. Furthermore, of 145 IF-negative samples, 38 155 (26%) were positive by both in-house and r-gene™ assays with 100% concordance between 156 the two tests (ten positive for influenza A, seven for influenza B and 21 for RSV). No PCR 157 inhibitor was detected. These various results indicate that the ability of the in-house triplex 158 assay to detect viral genomes was similar to that of the individual Flu-A/B and RSV-A/B r-159 gene™ assays. The assay was initially designed to detect current epidemic H1N1 and H3N2 160 viral strains. Thus, its sensitivity was re-assessed when the pandemic H1N1(2009) strain 161 emerged. Twenty-five clinical isolates, positive for the H1N1(2009) virus with the H1 and N1 162 subtype-specific RT-PCR assays, were provided by the Influenza National Reference Centre. 163 All of these samples were positive with the triplex assay, with a range of CTs similar to those 164 of samples positive for epidemic H1N1 strains. 165 -43 to 2009-10, samples were routinely collected from patients at Hôpital 169 Ambroise Paré and tested with the in-house triplex assay for the genomes of influenza A, 170 influenza B and RSV (table

  283 influenza A genomes detected, 175 were further characterized with the H1 and N1 187 subtype-specific RT-PCR assays. All 283 were A(H1N1) 2009 virus. No influenza B 188 infection was detected during the period. 189 triplex PCR for influenza A/B and RSV Papillard-Marechal et al DISCUSSION 190

  triplex PCR for influenza A/B and RSV Papillard-Marechal et al 10 daily practice, the procedure was simplified by adapting the extraction step to the MagNA 215

  triplex PCR for influenza A/B and RSV Papillard-Marechal et al 11

a

  FIGURE LEGENDS 367

Table 1 353Table 1 : Viral strains used to assess sensitivity and efficiency of the assay

 11

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Journal of Medical Virology

This work received financial support from the Assistance Publique -Hôpitaux de Paris.


MD Silvio ; Danese
email: sdanese@hotmail.com

Jean-Frédéric Colombel

Walter Reinisch

Paul Rutgeerts

Infliximab for Crohn's disease treatment: shifting therapeutic strategies after 10 years of clinical experience

Keywords: Inflammatory bowel disease < Disease-based, Crohn's disease < Disease-based, Immunosuppression < Topics, Biologics (IBD) < infliximab, Crohn's disease, treatment algorithm

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Introduction

Crohn's disease (CD) is a progressive disease that can be subdivided into 3 phenotypes: inflammatory, stricturing and penetrating. [START_REF] Sachar | Proposed classification of patient subgroups in Crohn's disease[END_REF] Initially, inflammatory disease is present in the majority of patients with CD. Over time, however, most patients will develop a penetrating or stricturing complication, owing to uncontrolled inflammation. [START_REF] Cosnes | Long-term evolution of disease behavior of Crohn's disease[END_REF] Within 20 years, 88% of patients will experience either stricturing (18%) or penetrating fistulising disease (70%). [START_REF] Cosnes | Long-term evolution of disease behavior of Crohn's disease[END_REF] The course of CD is heterogeneous and the progression of the disease varies considerably between patients. Many patients (43%) suffer a very severe initial flare and then experience few symptoms over the next 10 years. [START_REF] Solberg | Clinical course in Crohn's disease: results of a Norwegian population-based ten-year follow-up study[END_REF] The majority of patients (51%), however, experience chronic continuous (19%) or relapsing/remitting symptoms (32%). [START_REF] Solberg | Clinical course in Crohn's disease: results of a Norwegian population-based ten-year follow-up study[END_REF] In an inception cohort of 373 patients with CD, Munkholm et al. showed that many patients (45%) with active CD in the initial years continued to have chronically active disease over subsequent years (follow-up 8 years). [START_REF] Munkholm | Disease activity courses in a regional cohort of Crohn's[END_REF] Fifty percent had a chronic remitting disease course, while only 5% had inactive disease after 8 years.

The ultimate treatment goal in the management of CD should be to strive to change the underlying course of CD and restore normal bowel function. This requires the suppression of the underlying inflammation and the induction of complete mucosal healing. Mucosal healing has been associated with a reduction in serious complications (hospitalisation and surgery) and is an attainable goal with appropriate treatment. [START_REF] Rutgeerts | Comparison of scheduled and episodic treatment strategies of infliximab in Crohn's disease[END_REF][START_REF] Schnitzler | Mucosal healing predicts long-term outcome for maintenance therapy with infliximab in Crohn's disease[END_REF][START_REF] Solberg | Mucosal healing after initial treatment may be a prognostic marker for long-term outcome in inflammatory bowel disease[END_REF]

When to start anti-TNF therapy

There is a potential window of opportunity to influence the long-term evolution of CD early in the disease course when it is primarily inflammatory. Anti-TNF therapy is indicated in steroidrefractory, steroid-dependent and/or immunomodulator-refractory luminal CD and in patients intolerant to these conventional therapies. Additionally, anti-TNF therapy in conjunction with surgical drainage is indicated in complex fistulas in CD. Whether combination therapy improves efficacy is a key question, which may depend on the patient population. The risks of combined immunosuppression should be considered, especially in children, young adults, or the elderly. [START_REF] Haens | The London Position Statement of the World Congress of Gastroenterology on Biological Therapy for IBD With the European Crohn's and Colitis Organization: When to Start, When to Stop, Which Drug to Choose, and How to Predict Response?[END_REF] The thiopurine immunomodulators AZA and 6-mercaptopurine (6-MP) are frequently prescribed for patients in whom first-line therapies fail-in particular, those who are dependent on or do not have a response to systemic corticosteroids. [START_REF] Travis | European evidence based consensus on the diagnosis and management of Crohn's disease: current management[END_REF][START_REF] Lichtenstein | Management of Crohn's disease in adults[END_REF][START_REF] Lichtenstein | American Gastroenterological Association Institute technical review on corticosteroids, immunomodulators, and infliximab in inflammatory bowel disease[END_REF] When starting biological therapy in patients with CD naïve to thiopurines the combination of an anti-TNF agent and AZA is better for induction of remission and mucosal healing over 1 year. Whether combination therapy could improve outcomes from each of the anti-TNF agents remains unknown. [START_REF] Haens | The London Position Statement of the World Congress of Gastroenterology on Biological Therapy for IBD With the European Crohn's and Colitis Organization: When to Start, When to Stop, Which Drug to Choose, and How to Predict Response?[END_REF] Two anti-TNF therapies are available for treatment of patients with CD (Table 1). [START_REF]Remicade ® (infliximab) Summary of Product Characteristics (SPC)[END_REF][START_REF]Humira ® (adalimumab) Summary of Product Characteristics (SPC)[END_REF] Infliximab (intravenous infusion) was licensed for use in CD in the United States in 1998 and in Europe in 1999. Current use of infliximab has evolved based on evidence from clinical trials. Furthermore, infliximab has the broadest inflammatory bowel disease (IBD) label available in Europe and is approved for adult luminal and fistulising CD, paediatric luminal CD and adult ulcerative colitis (UC). [START_REF]Remicade ® (infliximab) Summary of Product Characteristics (SPC)[END_REF] Adalimumab (subcutaneous injection) was also licensed for use in CD in Europe and in the United States in 2007.

Over the past decade, the efficacy of anti-TNF has been demonstrated in various patient groups, including patients with different disease durations and previous exposure to conventional therapies. [START_REF] Rutgeerts | Comparison of scheduled and episodic treatment strategies of infliximab in Crohn's disease[END_REF][START_REF] Targan | A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor α for Crohn's disease[END_REF][START_REF] Lèmann | Infliximab plus azathioprine for steroid-dependent Crohn's disease patients: a randomised, placebo-controlled trial[END_REF][START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF][START_REF] Haens | Early combined immunosuppression or conventional management in patients with newly diagnosed Crohn's disease: an open randomised trial[END_REF][START_REF] Hyams | Induction and maintenance infliximab therapy for the treatment of moderate-to-severe Crohn's disease in children[END_REF][START_REF] Feagan | A randomized trial of methotrexate (MTX) in combination with infliximab (IFX) for the treatment of Crohn's disease (CD) [late-breaking abstract[END_REF][START_REF] Hanauer | Human anti-tumor necrosis factor monoclonal antibody (adalimumab) in Crohn's disease: the CLASSIC-I trial[END_REF][START_REF] Colombel | Adalimumab for maintenance of clinical response and remission in patients with Crohn's disease: the CHARM trial[END_REF][START_REF] Feagan | Effects of adalimumab therapy on incidence of hospitalization and surgery in Crohn's disease: results from the CHARM study[END_REF][START_REF] Rutgeerts | Adalimumab induces and maintains mucosal healing in patients with moderate to severe ileocolonic Crohn's disease-first results of the EXTEND Trial[END_REF] The spectrum of anti-TNF trials has provided lessons on an optimised way to treat patients with CD and raised the threshold for treatment goals. It is now clear that CD treatment must go beyond simply providing symptomatic control and aim to change the course of the disease. The anti-TNF clinical trial data have raised awareness and expectations of what treatment goals can be achieved (Table 2). [START_REF] Panaccione | Review article: treatment algorithms to maximize remission and minimize corticosteroid dependence in patients with inflammatory bowel disease[END_REF][START_REF] Steinhart | Induction therapy in Crohn's disease[END_REF] Mucosal healing is becoming an increasingly important parameter, as in some studies it has been shown to be linked with reductions in hospitalisations and surgeries as well as long-term remission. Achieving these goals will require rapid and sustained control of inflammation and appropriate management with earlier, more intensive use of biologic therapy in most patients. to AZA alone in immunomodulator-naïve patients. [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] SONIC also showed that the best results were achieved in patients with a high inflammatory burden (high C-reactive protein [CRP] and/or mucosal lesions) at baseline. [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] This trial is a landmark paper for CD management and has profound therapeutic implications for clinical practice.

Practical matters, such as patient preference regarding the mode of administration, may play a role in the selection of adalimumab as initial anti-TNF therapy. [START_REF] Yun | Selecting appropriate anti-TNF agents in inflammatory bowel disease[END_REF] The current European Crohn's & Colitis Organisation (ECCO) consensus in CD management states that 'All currently available anti-TNF therapies appear to have similar efficacy and adverse-event profiles, so the choice depends on availability, route of delivery, patient preference, cost and national guidance'. [START_REF] Dignass | The second European evidence-based consensus on the diagnosis and management of Crohn's disease: current management[END_REF] A qualitative study of patient preferences of anti-TNF agents in rheumatoid arthritis suggests that younger patients are more confident about self-administering treatment and slightly prefer the convenience of subcutaneous dosing, whereas older patients prefer the perceived safety of infusion in a clinic. [START_REF] Chilton | Treatment choices, preferences and decision-making by patients with rheumatoid arthritis[END_REF] Decisions should be made on an individual basis and consider the preferences of both the patient and physician. [START_REF] Lichtenstein | Management of Crohn's disease in adults[END_REF][START_REF] Dignass | The second European evidence-based consensus on the diagnosis and management of Crohn's disease: current management[END_REF] This paper will review the infliximab clinical trial data and introduce an evidence-based infliximab treatment algorithm for the management of luminal and complex fistulising CD, based on the clinical data and expert opinion, and will discuss the clinical and practical implications of the SONIC trial in the daily management of CD.

Identifying patients with progressive disease (including fistulising disease)

The treatment of patients with CD should be customised according to factors that predict progressive disease. The European Crohn's and Colitis Organization (ECCO) consensus on the management of CD recognises that the course of CD may be predicted by clinical factors at diagnosis (including age and perianal disease), which should be taken into account when determining the initial therapeutic strategy. [START_REF] Dignass | The second European evidence-based consensus on the diagnosis and management of Crohn's disease: current management[END_REF] Several studies have confirmed that young age at diagnosis (<40 years old) and perianal disease are associated with poor outcomes/disabling disease. [START_REF] Beaugerie | Predictors of Crohn's disease[END_REF][START_REF] Franchimont | Clinical pattern of corticosteroid dependent Crohn's disease[END_REF][START_REF] Loly | Predictors of severe Crohn's disease[END_REF] Perianal fistulas are the most common type of fistula. [START_REF] Lapidus | Clinical course of colorectal Crohn's disease: a 35-year follow up study of 507 patients[END_REF] Perianal fistulas are a therapeutic challenge and are associated with decreased QoL and increased risk of total colectomy. [START_REF] Lapidus | Clinical course of colorectal Crohn's disease: a 35-year follow up study of 507 patients[END_REF][START_REF] Lichtenstein | Infliximab maintenance treatment reduces hospitalizations, surgeries, and procedures in fistulizing Crohn's disease[END_REF] Perianal fistulas are classified as either simple or complex. Simple fistulas are superficial, with a single external opening, with no pain or fluctuation suggesting abscess, rectovaginal fistula or anorectal stricture. All other fistulas, including active rectal disease, are considered complex. [START_REF] Sandborn | American Gastroenterological Association Clinical Practice Committee. AGA technical review on perianal Crohn's disease[END_REF]

Treating complex fistulising CD

Infliximab clinical data

The ACCENT II (A Crohn's disease Clinical trial Evaluating infliximab in a New Long term Treatment regimen in patients with fistulising Crohn's disease) trial demonstrated that infliximab is effective in treating fistulising CD. In this double-blind, placebo-controlled trial, 306 adult patients with CD and one or more draining abdominal or perianal fistulas of at least 3 months' duration were randomised to receive a 3-dose induction regimen of infliximab 5 mg/kg at weeks 0, 2 and 6. Sixtynine percent of patients (195/282) responded (50% reduction in draining fistulas) to infliximab. At week 14, these responders were randomly assigned to receive placebo maintenance (n=99) or infliximab maintenance (n=96). The remaining initial nonresponders (n=87) were randomly assigned to receive maintenance therapy with infliximab (n=43) or placebo (n=44) until week 54. [START_REF] Sands | Infliximab maintenance therapy for fistulizing Crohn's disease[END_REF] From week 22, patients receiving placebo who experienced a loss of response were eligible to cross over to treatment with infliximab 5 mg/kg, and patients in the infliximab 5 mg/kg group could cross over to treatment with infliximab 10 mg/kg. [START_REF] Sands | Infliximab maintenance therapy for fistulizing Crohn's disease[END_REF] The primary analysis was the time to loss of response among patients who responded at week 14 and underwent randomisation. [START_REF] Sands | Infliximab maintenance therapy for fistulizing Crohn's disease[END_REF] Among responders, those receiving infliximab maintenance therapy had a significantly longer time to loss of response than those receiving placebo. The median time to loss of response was 14 Overall, 62% of placebo patients had a loss of response compared with 42% of patients receiving infliximab. [START_REF] Sands | Infliximab maintenance therapy for fistulizing Crohn's disease[END_REF] At week 54, significantly more responders in the infliximab maintenance group had a fistula response compared with placebo (46% vs 23%, P=0.001). A similar level of efficacy was observed in patients who had a complete response (absence of draining fistulas) of patients in the infliximab group (36% vs 23%, P=0.009). [START_REF] Sands | Infliximab maintenance therapy for fistulizing Crohn's disease[END_REF] Reduced hospitalisation/surgery rates ACCENT II also demonstrated that infliximab significantly reduces the rate of hospitalisation and surgery in patients with fistulising CD. Patients receiving maintenance infliximab were more than twice less likely to be hospitalised due to fistulising disease than those receiving placebo (8.6% vs 18.9% [all randomised patients] and 7.3% vs 18.2% [week 14 responders], P<0.05 for both). [START_REF] Lichtenstein | Infliximab maintenance treatment reduces hospitalizations, surgeries, and procedures in fistulizing Crohn's disease[END_REF] Importantly, scheduled infliximab treatment resulted in a 50% reduction in the mean number of all surgeries and procedures (eg, resection of the bowel, fistula-related surgeries, ostomy placement/revision) compared with placebo for all randomised patients (P<0.01) and patients randomised as responders (P<0.05). [START_REF] Lichtenstein | Infliximab maintenance treatment reduces hospitalizations, surgeries, and procedures in fistulizing Crohn's disease[END_REF] While the cumulative number of surgeries and procedures for patients randomised as responders during the study increased at a relatively slow rate in the infliximab group, the increase in the placebo group was markedly greater. [START_REF] Lichtenstein | Infliximab maintenance treatment reduces hospitalizations, surgeries, and procedures in fistulizing Crohn's disease[END_REF] Evidence-based treatment recommendation ACCENT II provides clear evidence of the effectiveness of infliximab in treating fistulising CD.

Based on those data and clinical experience, patients with complex fistulising CD should start treatment with infliximab with or without 2-2.5 mg/kg AZA/6MP immediately (Figure 1). Prior surgical intervention should be considered, with potential abscess drainage and seton placement. Early in the course of luminal CD, patients should be stratified according to their risk of progressive disease. Unfortunately, there are no consistent criteria across studies and more work needs to be done to establish clinical, serological and genetic predictors for progressive disease. Beaugerie et al identified factors at diagnosis predictive of a subsequent 5-year disabling CD course. This study defined 'disabling disease' as the presence of at least one of the following clinical severity criteria: ≥2 steroid courses, steroid dependence, hospitalisation, chronic (>12 months) symptoms, need for immunosuppressants or need for surgery. [START_REF] Beaugerie | Predictors of Crohn's disease[END_REF] Of the 1123 patients with 5-year follow-up data, the rate of disabling disease was 85.2%. [START_REF] Beaugerie | Predictors of Crohn's disease[END_REF] In a prospective validation cohort of 302 patients, 3 independent risk factors for disabling disease course were identified: initial steroid requirement, age <40 years and the presence of perianal disease. [START_REF] Beaugerie | Predictors of Crohn's disease[END_REF] Patients with ≥2 risk factors have been shown to have a high likelihood of a disabling course. [START_REF] Beaugerie | Predictors of Crohn's disease[END_REF] Although this study defined disabling disease, no consistent definition for disabling disease has been adopted from many other studies.

Stratifying patients with luminal CD based on risk factors

For example, the study by Beaugerie et al. included need for immunosuppression as disabling disease, whereas in other studies, this term was referred to was steroid dependency, [START_REF] Franchimont | Clinical pattern of corticosteroid dependent Crohn's disease[END_REF] stenosis/obstruction [START_REF] Lichtenstein | Factors associated with the development of intestinal strictures or obstructions in patients with Crohn's disease[END_REF] or mortality. [START_REF] Munkholm | Intestinal cancer risk and mortality in patients with Crohn's disease[END_REF] Additional risk factors for progressive disease in luminal CD can be found in Table 3. From clinical experience, patients with the following characteristics should also be considered as those at risk for progressive disease: extensive small bowel disease, severe upper GI disease, severe rectal disease, younger age and perianal lesions.

Treating luminal CD

Historically, the treatment of luminal CD has taken a sequential step-up approach that involves starting therapy with the least toxic drug and adding in other drugs if there is no response. In practice, this means starting with 5-ASA and then progressing to corticosteroids, immunosuppressants, anti-TNF therapy and finally surgery. The use of infliximab tends to be limited to patients with refractory CD, steroid-resistant patients or patients not responding to immunosuppressants. [START_REF] Panaccione | Review article: treatment algorithms to maximize remission and minimize corticosteroid dependence in patients with inflammatory bowel disease[END_REF] Many patients receiving conventional therapy, however, remain on drugs with low efficacy for long periods and continue to have active disease. This uncontrolled inflammation can often lead to mucosal damage. It is becoming clear that conventional therapy may not be the optimal approach as there are important limitations to consider. Corticosteroids may also control symptoms on the short term; long-term outcomes are less favourable and discouraged. [START_REF] Panaccione | Review article: treatment algorithms to maximize remission and minimize corticosteroid dependence in patients with inflammatory bowel disease[END_REF] Significantly, corticosteroids are not effective in maintaining remission; only 25% of patients taking corticosteroids will be in remission after a year, even if given with immunomodulators. [START_REF] Dignass | The second European evidence-based consensus on the diagnosis and management of Crohn's disease: current management[END_REF] Corticosteroids are also not effective for inducing mucosal healing. In addition, long-term corticosteroid use is associated with serious side effects, such as weight gain, cataracts, hyperglycaemia, osteoporosis and increased risk of infection. Consequently, it is important to limit the use of corticosteroids [START_REF] Dignass | The second European evidence-based consensus on the diagnosis and management of Crohn's disease: current management[END_REF] and avoid repeated cycles of these drugs. Azathioprine (AZA) may be used as adjunctive or steroid-sparing therapy in some patients; however, its slow onset of action prevents it from being used as monotherapy in active CD. [START_REF] Dignass | The second European evidence-based consensus on the diagnosis and management of Crohn's disease: current management[END_REF] Additionally, its effect on the mucosa is limited.

There is now a body of evidence demonstrating that earlier use of immunosuppressants and antitumour necrosis factor (TNF) therapy improves clinical outcomes. This has been clearly demonstrated with infliximab trials. It is becoming accepted that an accelerated step-up approach is needed in patients with moderately or severely active luminal CD. The earlier use of immunosuppressants and anti TNF therapy will induce and maintain remission, reduce steroid use and promote mucosal healing. Achieving this will provide the opportunity to impact the natural history of CD and thus reduce the risk of serious complications, such as hospitalisation and surgery.

An alternative to drugs could be surgery in patients with limited ileo-cecal CD. Understanding the best strategy will be clarified by the randomized-controlled study now undergoing in The Netherlands; the Laparoscopic Ileocolic Resection Versus Infliximab Treatment of Distal Ileitis in Crohn's Disease (LIR!C) trial. Once this study is completed, it will provide information if surgery will lead to avoiding the use of medication and longer symptom-free efficacy than anti-TNF therapies.

The results of this trial can be expected in 2012 or 2013.

Infliximab clinical data in luminal CD

The ACCENT I trial demonstrated the efficacy of infliximab in luminal CD. In this study, 573 patients with active CD (CDAI 220-400) received a single infusion of infliximab 5 mg/kg. After the initial infusion, 58% of patients responded to infliximab. [START_REF] Hanauer | Maintenance infliximab for Crohn's disease: the ACCENT I randomised trial[END_REF] At week 2, patients responding to infliximab were randomised to receive either episodic (infliximab 5 mg/kg infusion followed by placebo infusions at weeks 0, 2 and 6 and then every 8 weeks) or scheduled treatment (infliximab 5 mg or 10 mg/kg at weeks 0, 2 and 6 and then every 8 weeks). [START_REF] Hanauer | Maintenance infliximab for Crohn's disease: the ACCENT I randomised trial[END_REF] At 1 year, over 3 times as many patients receiving scheduled infliximab (29%) were in steroid-free remission compared with those receiving episodic infliximab (9%) (P=0.004). [START_REF] Hanauer | Maintenance infliximab for Crohn's disease: the ACCENT I randomised trial[END_REF] A sustained clinical response was more likely with scheduled rather than episodic treatment. [START_REF] Hanauer | Maintenance infliximab for Crohn's disease: the ACCENT I randomised trial[END_REF] ACCENT I showed that scheduled infliximab treatment every 8 weeks is more effective than episodic treatment and formed the basis for infliximab dosing.

In addition, scheduled infliximab was associated with fewer hospitalisations and higher rates of mucosal healing. Furthermore, ACCENT I demonstrated the efficacy of infliximab dose escalation. Among patients receiving infliximab 5 mg/kg scheduled treatment who lost response, approximately 90% re-established response after receiving 10 mg/kg. Approximately 80% of patients who lost response while in the 10 mg/kg scheduled strategy group achieved response after receiving 15 mg/kg. [START_REF] Rutgeerts | Comparison of scheduled and episodic treatment strategies of infliximab in Crohn's disease[END_REF] The GETAID (Groupe d'Etude Therapeutique des Affections Inflammatoires Digestives) study also demonstrated the benefits of initiating infliximab treatment earlier by showing that infliximab plus AZA combination therapy is more effective that AZA monotherapy in AZA-naïve patients. In the GETAID trial, 113 steroid dependent patients with active CD were stratified into 2 groups: AZA/6-MP failures and AZA/6-MP-naïve patients. [START_REF] Lèmann | Infliximab plus azathioprine for steroid-dependent Crohn's disease patients: a randomised, placebo-controlled trial[END_REF] Patients were randomised to infliximab 5 mg/kg or placebo at weeks 0, 2 and 6, with no maintenance treatment. All patients were treated with stable infliximab plus AZA/6-MP compared with patients receiving AZA/6-MP alone were in steroid-free clinical remission at week 12 (75% vs 38%; P<0.001) and week 24 (57% vs 29%; P=0.003). [START_REF] Lèmann | Infliximab plus azathioprine for steroid-dependent Crohn's disease patients: a randomised, placebo-controlled trial[END_REF] The effect of infliximab on clinical remission was greater in AZA-naïve patients than AZA-failures, suggesting that earlier infliximab use may be beneficial in CD patients.

Importantly, the GETAID study also indicated that the proportion of patients receiving AZA who achieved steroid-free remission at week 12 declined over the course of the study, demonstrating that there was no 'bridging effect' and confirming that infliximab should not be used as a bridge to immunomodulator therapy in this patient population. This clearly shows that AZA cannot sustain the efficacy induced by infliximab, and these patients are more likely to benefit from scheduled infliximab maintenance therapy. [START_REF] Lèmann | Infliximab plus azathioprine for steroid-dependent Crohn's disease patients: a randomised, placebo-controlled trial[END_REF] Additionally, Treton et al assessed the impact of AZA withdrawal after long-term remission with AZA treatment (median duration of 68.4 months) in a cohort of 66 patients. The primary end point was clinical relapse, and the study found that AZA withdrawal was associated with a high rate of relapse regardless of remission duration under treatment. [START_REF] Treton | Azathioprine withdrawal in patients with Crohn's disease maintained on prolonged remission: a high risk of relapse[END_REF] An important question is whether it is necessary to continue with infliximab plus AZA combination therapy or can AZA be withdrawn? The influence of immunosuppressive withdrawal in patients in remission with combination therapy has been assessed in an open-label, randomised, controlled study. Patients with controlled disease (>6 months) on infliximab (5 mg/kg) plus immunosuppressives were randomised to continue or discontinue immunosuppressives. All patients received scheduled infliximab maintenance therapy for 104 weeks. [START_REF] Van Assche | Withdrawal of immunosuppression in Crohn's disease treated with scheduled infliximab maintenance: a randomized trial[END_REF] The study found that the continuation of immunosuppressives beyond 6 months provided no clear benefit over scheduled infliximab monotherapy. [START_REF] Van Assche | Withdrawal of immunosuppression in Crohn's disease treated with scheduled infliximab maintenance: a randomized trial[END_REF] This is supported by Lichtenstein et al. in a study that reviewed the effect of concomitant immunomodulator and infliximab maintenance therapy using data from the ACCENT I and ACCENT II trials. Similarly, this study also found that use of concomitant immunomodulators did not improve efficacy in patients receiving maintenance [START_REF] Lichtenstein | Clinical trial: benefits and risks of immunomodulators and maintenance infliximab for IBD-subgroup analyses across four randomized trials[END_REF] In contrast to these studies, a recent study assessed the effect of concomitant use of immunosuppressives (AZA or methotrexate [MTX]) with scheduled infliximab treatment in patients with IBD over semester time periods. A semester was defined as a 6-month period of infliximab treatment. The study found that patients with IBD who received combination therapy with an immunosuppressive had reduced IBD activity, reduced infliximab dose escalation and less need to switch to another biologic. [START_REF] Sokol | Usefulness of co-treatment with immunomodulators in patients with inflammatory bowel disease treated with scheduled infliximab maintenance therapy[END_REF] Currently, the mechanism of superiority of combination therapy is unclear. Possible explanations may include a pharmacokinetic or additive effect. Further studies are still needed to help clarify this as well as questions on how long to treat patients with combined immunosuppressive therapy and when to discontinue AZA.

Evidence-based treatment recommendation

Patients with luminal CD and risk factors for progressive disease should be monitored closely.

These patients require aggressive intervention and may benefit from accelerated treatment with early use of anti-TNF therapy +/-2-2.5 mg/kg AZA.

The recommended treatment pathway for patients with luminal CD with risk factors is shown in the second arm of the algorithm in Figure 2. All patients should initially receive steroids plus AZA/6-MP. Although there is no evidence in adults to support this initial treatment regimen, this approach has been very successful in paediatric CD. [START_REF] Markowitz | A multicenter trial of 6-mercaptopurine and prednisone in children with newly diagnosed Crohn's disease[END_REF] If remission is achieved, steroids should be tapered and AZA monotherapy continued. If the patient then experiences a delayed relapse (after 6 months), steroids should be reintroduced and the response assessed after 4 weeks. If the patient experiences an early relapse (within 6 months) after initial remission with steroids plus AZA/ 6-MP, then infliximab plus AZA/6-MP therapy should be initiated.

If remission is not achieved using steroids plus AZA/6-MP (within 4 weeks), then treatment with infliximab +/-AZA/6-MP should be initiated. After 6 to 12 months of stable remission (normal CRP, steroid-free clinical remission with mucosal healing), stepping down to infliximab monotherapy may be an option for some patients. Disease) trial is a landmark trial for the management of luminal CD, demonstrating the benefits of an infliximab-based treatment strategy. [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] The findings from SONIC are changing the way luminal CD is treated in clinical practice and have also impacted treatment guidelines such as ECCO and European Panel on the Appropriateness of Crohn's Disease Treatment (EPACT). Patients (n=508) with early, moderate to severe CD and naïve to immunomodulators and biologics were randomised to receive AZA 2.5-mg/kg capsules plus placebo infusions, infliximab 5-mg/kg infusions (week 0, 2 and 6 and then every 8 weeks) plus placebo capsules or infliximab 5-mg/kg infusions plus AZA 2.5-mg/kg capsules for 54 weeks. [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] The primary end point was steroid-free remission at week 26.

Endoscopy was performed at weeks 0 and 26.

An important finding of the SONIC trial was that infliximab was superior to AZA monotherapy in inducing steroid-free remission. Significantly more patients receiving infliximab monotherapy (44.4%) or infliximab plus AZA combination therapy (56.8%) were in steroid-free clinical remission compared with patients receiving AZA monotherapy (30.6%) at week 26 (IFX monotherapy vs AZA monotherapy, P=0.009; IFX+AZA vs AZA monotherapy, P<0.001). [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] Interestingly, in this study, infliximab combination therapy was more effective in inducing steroid-free clinical remission than infliximab monotherapy (P=0.022). These effects were sustained through to week 50. [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] SONIC demonstrated that an infliximab-based treatment strategy is more effective than AZA monotherapy in AZA-naïve patients.

Another important finding from SONIC was the superiority of infliximab over AZA monotherapy in inducing mucosal healing. More patients experienced complete mucosal healing while receiving infliximab monotherapy (30%; P=0.023) or infliximab-plus-AZA combination therapy (44%; P<0.001) than AZA monotherapy (17%). [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] Although SONIC did not assess impact on outcomes SONIC also revealed that patients with high inflammatory burden (high CRP and/or endoscopic lesions) at baseline derived the greatest benefit from an infliximab-based treatment strategy. In patients with high baseline CRP levels (≥0.8 mg/dL), significantly more were in steroid-free clinical remission with infliximab plus AZA combination therapy (63.5%, P<0.001) or infliximab monotherapy (47.5%, P=0.004) than AZA monotherapy (27.6%). [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] Superiority of infliximab over AZA monotherapy was also observed in patients with mucosal lesions at baseline but not in patients with no lesions at baseline. [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] This effect was even greater in patients with both high CRP and mucosal lesions at baseline, with 68.8% of patients receiving infliximab plus AZA combination therapy and 56.9% receiving infliximab monotherapy in clinical remission at week 26 compared with 28% (P<0.001) of AZA monotherapy-treated patients. [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] The group treated with early combined immunosuppressive therapy at the end of the study period had superior mucosal healing, thus confirming the capacity of infliximab to significantly heal the intestinal mucosa in CD.

Evidence-based treatment recommendation

SONIC has shown that anti-TNF therapy should be initiated much earlier in patients with luminal CD. The recommended treatment pathway for patients with luminal CD (no risk factors) is shown in the third arm of the algorithm in Figure 3. Initial treatment should be with steroids and if remission is achieved (top arm), then steroids should be tapered down until they are discontinued. If there is a delayed relapse (after 6 months) after discontinuing steroids, then the patient should be retreated with steroids and the response assessed at 4 weeks. If the patient experiences an early relapse (within 6 months) after discontinuing steroids, there are 2 options to consider: 1) initiate infliximab +/-2-2.5 mg/kg AZA/6-MP if the patient has lesions and/or elevated CRP or 2) initiate steroids plus 2-2.5 mg/kg AZA/6-MP combination therapy and then if the patient relapses move on to infliximab +/-2-2.5 mg/kg AZA/6-MP (moving down to lower arm).

If remission is not achieved within 4 weeks of initial steroid therapy (lower arm), infliximab +/-AZA/6-MP should be initiated. In either arm, after 6 to 12 months of stable remission (normal CRP, steroid-free clinical remission with mucosal healing), stepping down to infliximab monotherapy may be an option for some patients. Infliximab should be considered as monotherapy in elderly patients.

Infliximab monotherapy rather than combination therapy, however, should be given to young males with CD owing to the risk of hepatosplenic T-cell lymphoma. Although this condition is extremely rare, it is very serious, with most cases proving fatal. Thirty-six cases have been reported in which 20 were treated with infliximab plus a thiopurine and 16 were on thiopurine monotherapy. Four cases that included infliximab and thiopurine also took adalimumab. One case was exposed to infliximab, adalimumab and natalizumab. Of the 31 patients whose gender was known, only 2 were female. [START_REF]Data on file[END_REF] It is prudent, therefore, not to administer infliximab plus AZA combination in young male

Loss of response and treatment failure

Some patients with CD may experience loss of response over time and/or develop intolerance to infliximab. It is estimated that this might occur in about 10% of treated patients per year. [START_REF] Gisbert | Loss of response and requirement of infliximab dose intensification in Crohn's disease: a review[END_REF] Patients with diminished or loss of response to infliximab therapy may respond to optimized dosing regimens of the same agent or switching to another agent. [START_REF] Haens | The London Position Statement of the World Congress of Gastroenterology on Biological Therapy for IBD With the European Crohn's and Colitis Organization: When to Start, When to Stop, Which Drug to Choose, and How to Predict Response?[END_REF] Before switching to another agent, one should consider optimizing their first agent if possible. Infliximab allows some dosing flexibility which has been demonstrated from experience in the Leuven cohort. [START_REF] Schnitzler | Long-term outcome of treatment with infliximab in 614 patients with Crohn's disease: results from a single-centre cohort[END_REF] Schnitzler et al assessed the long-term clinical benefits of infliximab in 614 consecutive patients with CD from a single centre in an observational study over a median of 4.6 years. Of 547 initial responders, approximately 50% needed an intervention. A reduction in the interval between infusions was needed in 108 patients (19.7%); an increase in the dose to 10 mg/kg and/or a re-induction with infliximab infusions at weeks 0, 2 and 6 was needed in 144 patients (26.3%; 63 patients with a re-induction and 89 patients with an increase in the dose of infliximab); and an increase in the dose plus a reduction in the interval was needed in only 21 patients (3.8%). Overall, 103 of the 144 patients (71.5%) with an increase in dose and/or a re-induction with infliximab could go back to the standard dose of 5 mg/kg and 61.9% of patients (13/21) with an increase in dose and a shortening of the interval could go back to 5 mg/kg and dosing at 8 week intervals. A total of 28.7% of patients with a shortened interval between infusions (31/108) could again extend the interval to 8 weeks. In the total cohort of initial responders, only 21.6% (n=118) had to stop infliximab because of loss of response despite interventions. [START_REF] Schnitzler | Long-term outcome of treatment with infliximab in 614 patients with Crohn's disease: results from a single-centre cohort[END_REF] When optimizing the first agent is not successful, then switching may be an option.

In a randomized, placebo-controlled trial (GAIN [Gauging Adalimumab Efficacy in Infliximab

Nonresponders]), switching to adalimumab was effective in patients with previous loss of response to their first anti-TNF therapy. At baseline, 48% (n=77) of patients in the adalimumab group had 18 versus 34% (56 of 166) of patients in the placebo group (P=0.001). [START_REF] Sandborn | Adalimumab induction therapy for Crohn disease previously treated with infliximab: a randomized trial[END_REF] Some patients with CD will not respond to their first anti-TNF treatment. Reports on this patient population are very limited. In a retrospective survey, Allez et al. observed that 12 out of 18 patients responded to a 3rd anti-TNF after primary failure to one or 2 prior anti-TNF treatment(s). [START_REF] Allez | The efficacy and safety of a third anti-TNF monoclonal antibody in Crohn's disease after failure of two other anti-TNF antibodies[END_REF] This treatment option requires thorough case-by-case discussion and should only be considered in patients with no other therapeutic options. [START_REF] Allez | The efficacy and safety of a third anti-TNF monoclonal antibody in Crohn's disease after failure of two other anti-TNF antibodies[END_REF]

When to stop treatment

To date, recommendations on when to stop anti-TNF therapy cannot be made due to insufficient data. [START_REF] Haens | The London Position Statement of the World Congress of Gastroenterology on Biological Therapy for IBD With the European Crohn's and Colitis Organization: When to Start, When to Stop, Which Drug to Choose, and How to Predict Response?[END_REF] Patients in stable remission may have no medical reason to stop anti-TNF therapy unless there are circumstances when cessation may be necessary-for example, if a patient is unwilling to continue treatment with the drug, in situations such as pregnancy or if changes in reimbursement affect coverage of treatment. Preliminary evidence suggests that some patients will remain in clinical remission for >1 year despite cessation of infliximab treatment. [START_REF] Haens | The London Position Statement of the World Congress of Gastroenterology on Biological Therapy for IBD With the European Crohn's and Colitis Organization: When to Start, When to Stop, Which Drug to Choose, and How to Predict Response?[END_REF] A retrospective study by Domenech et al, evaluated clinical outcome after a successful course of infliximab treatment for maintenance of response in both luminal and perianal CD. [START_REF] Domènech | Clinical evolution of luminal and perianal Crohn's disease after inducing remission with infliximab: how long should patients be treated?[END_REF] Infliximab discontinuation was successful for patients in patients with luminal CD treated for one year (69% cumulative probability of being free of relapse at 12 months). Conversely, patients with perianal disease demonstrated early relapse with only 34% (versus 83% in patients with luminal disease) maintaining remission at 1 year. Consequently, due to a high rate of early relapse, infliximab discontinuation is not recommended in perianal CD. [START_REF] Domènech | Clinical evolution of luminal and perianal Crohn's disease after inducing remission with infliximab: how long should patients be treated?[END_REF] The STORI (infliximab diSconTinuation in CrOhn's disease patients in stable Remission on combined therapy with Immunosuppressors) trial assessed the risk of relapse after discontinuation of infliximab in patients on combined maintenance therapy with immunosuppressors. [START_REF] Louis | Infliximab discontinuation in Crohn's disease patients in stable remission on combined therapy with immunosuppressors: interim analysis of a prospective cohort study[END_REF] Patients who received scheduled infliximab plus immunosuppressive combination therapy for at least 1 year and who were also in steroid-free remission for ≥6 months were included. [START_REF] Louis | Infliximab discontinuation in Crohn's disease patients in stable remission on combined therapy with immunosuppressors: interim analysis of a prospective cohort study[END_REF] More than 50% of patients had relapsed after 18 months of treatment discontinuation; however, patients who did relapse were successfully re-treated with [START_REF] Louis | Infliximab discontinuation in Crohn's disease patients in stable remission on combined therapy with immunosuppressors: a prospective ongoing cohort study[END_REF] These results show that routine discontinuation of infliximab therapy may lead to a very high disease relapse and could not be a sound treatment decision.

Managing fistulas

Patients with fistulising CD with acute suppurative fistulas must not initiate infliximab therapy until a source for possible infection, specifically abscess, has been excluded. Patients with fistulising CD who have responded to an induction regimen with anti-TNF therapy, should receive scheduled retreatment with infliximab or adalimumab, since this is effective for maintaining fistula closure or response. [START_REF] Haens | The London Position Statement of the World Congress of Gastroenterology on Biological Therapy for IBD With the European Crohn's and Colitis Organization: When to Start, When to Stop, Which Drug to Choose, and How to Predict Response?[END_REF] Combined medical and surgical strategies have evolved, with drainage of sepsis and insertion of a seton, followed by two doses of anti-TNF therapy, fistula curettage, and then further anti-TNF therapy. [START_REF] Haens | The London Position Statement of the World Congress of Gastroenterology on Biological Therapy for IBD With the European Crohn's and Colitis Organization: When to Start, When to Stop, Which Drug to Choose, and How to Predict Response?[END_REF][START_REF] Hyder | Fistulating anal Crohn's disease: results of combined surgical and infliximab treatment[END_REF][START_REF] Tougeron | Predicting factors of fistula healing and clinical remission after infliximab-based combined therapy for perianal fistulizing Crohn's disease[END_REF] Insertion of drainage seton sutures at the time of preinfliximab examination under anesthesia and removal after the second infusion are considered routine practice in this patient population. [START_REF] Hyder | Fistulating anal Crohn's disease: results of combined surgical and infliximab treatment[END_REF]

Pregnancy

Post-marketing reports from approximately 300 pregnancies exposed to infliximab, do not indicate unexpected effects on pregnancy outcome. Due to its inhibition of TNFα, infliximab administered during pregnancy could affect normal immune responses in the newborn. The available clinical experience is too limited to exclude a risk, and administration of infliximab is therefore not recommended during pregnancy. [START_REF]Remicade ® (infliximab) Summary of Product Characteristics (SPC)[END_REF]

Patient screening and vaccination

Patients taking TNF-blockers are more susceptible to serious infections. Tuberculosis, bacterial infections, including sepsis and pneumonia, invasive fungal infections, and other opportunistic infections have been observed in patients treated with infliximab. [START_REF]Remicade ® (infliximab) Summary of Product Characteristics (SPC)[END_REF] Before starting treatment with infliximab, all patients must be evaluated for both active and latent tuberculosis. [START_REF] Haens | The London Position Statement of the World Congress of Gastroenterology on Biological Therapy for IBD With the European Crohn's and Colitis Organization: When to Start, When to Stop, Which Drug to Choose, and How to Predict Response?[END_REF][START_REF]Remicade ® (infliximab) Summary of Product Characteristics (SPC)[END_REF] If active tuberculosis is diagnosed, infliximab therapy must not be initiated. No data are available on the response to vaccination with live vaccines or on the secondary transmission of infection by live

Safety Considerations

The safety findings observed in the SONIC trial demonstrate that the incidence of adverse events (including serious adverse events) and serious infections was similar among the infliximab monotherapy, infliximab plus AZA combination therapy and AZA monotherapy groups. However, infusion reactions occurred less frequently among patients receiving combination therapy than among those receiving infliximab monotherapy. [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] Increased risks of rare but serious toxic effects associated with combination therapy must be considered. [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] Additionally, increased relative risk of serious and opportunistic infections associated with concomitant use of corticosteroids as a third immunosuppressive agent must be taken into account. 62 The choice of infliximab monotherapy or combination therapy in patients who have not received such therapy previously is an individualized benefit-risk decision. [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF] Additionally, the benefit to risk profile must be considered when choosing an anti-TNF agent.

Conclusions

Ten years ago, the options available for patients with CD were limited. It was possible to achieve symptomatic remission with some improvements in QoL; however, therapy was restricted to episodic induction and the treatment of each individual disease flare. Today, with timely drug intervention, it is possible to induce clinical remission that can be sustained over the long term with scheduled maintenance therapy. Over 10 years of clinical data and experience demonstrate the excellent efficacy and safety profile of infliximab.

Based on the infliximab clinical trial data, it may now be possible to change the underlying course of CD and restore normal bowel function, thereby improving the patient's QoL. The evidence shows that there is a window of opportunity early in the course of CD when patients benefit most from infliximab therapy. The landmark SONIC trial has significantly impacted the way CD is treated Furthermore, there is clear evidence for infliximab as an option for treating fistulising CD, and patients with luminal CD with risk factors should be considered for accelerated step-up therapy.

A controlled clinical trial demonstrating how the treatment approach of early intervention from the SONIC trial can be applied to all of the anti-TNF therapies is warranted. The early use of an evidence-based infliximab treatment algorithm, such as the one proposed in this paper, will significantly improve outcomes for patients with CD (Figure 4). 2. Therapeutic goals in CD [START_REF] Panaccione | Review article: treatment algorithms to maximize remission and minimize corticosteroid dependence in patients with inflammatory bowel disease[END_REF][START_REF] Steinhart | Induction therapy in Crohn's disease[END_REF] • Induce rapid response and maintain steroid-free remission [START_REF] Panaccione | Review article: treatment algorithms to maximize remission and minimize corticosteroid dependence in patients with inflammatory bowel disease[END_REF] • Achieve and maintain complete mucosal healing [START_REF] Panaccione | Review article: treatment algorithms to maximize remission and minimize corticosteroid dependence in patients with inflammatory bowel disease[END_REF] • Improve quality of life (QoL) [START_REF] Panaccione | Review article: treatment algorithms to maximize remission and minimize corticosteroid dependence in patients with inflammatory bowel disease[END_REF] • Avoid complications (ie, hospitalisation and surgery) [START_REF] Panaccione | Review article: treatment algorithms to maximize remission and minimize corticosteroid dependence in patients with inflammatory bowel disease[END_REF] • Prevent disease-related mortality [START_REF] Panaccione | Review article: treatment algorithms to maximize remission and minimize corticosteroid dependence in patients with inflammatory bowel disease[END_REF] • Avoid treatment-related mortality [START_REF] Panaccione | Review article: treatment algorithms to maximize remission and minimize corticosteroid dependence in patients with inflammatory bowel disease[END_REF] • Stricturing, penetrating behavior [START_REF] Solberg | Clinical course in Crohn's disease: results of a Norwegian population-based ten-year follow-up study[END_REF] • Smoking [START_REF] Sutherland | Effect of cigarette smoking on recurrence of Crohn's disease[END_REF][START_REF] Lindberg | Smoking in Crohn's disease: effect on localisation and clinical course[END_REF] • Serologic markers [START_REF] Reese | Diagnostic precision of anti-Saccharomyces cerevisiae antibodies and perinuclear antineutrophil cytoplasmic antibodies in inflammatory bowel disease[END_REF] -ASCA/pANCA

F

• Genetic markers [START_REF] Gearry | Effect of inflammatory bowel disease classification changes on NOD2 genotype-phenotype associations in a population-based cohort[END_REF][START_REF] Armuzzi | Genotype-phenotype analysis of the Crohn's disease susceptibility haplotype on chromosome 5q31[END_REF] -NOD2/IBD5

• Elevated CRP [START_REF] Boirivant | The clinical significance of serum C reactive protein levels in Crohn's disease. Results of a prospective longitudinal study[END_REF] • Severe endoscopic lesions (deep ulcerations) [START_REF] Allez | Long term outcome of patients with active Crohn's disease exhibiting extensive and deep ulcerations at colonoscopy[END_REF]

Complex

  (Study Of biologic and immunomodulator-Naive patients In Crohn's disease) was conducted in AZA-naïve patients with moderate to severe CD (mean duration 2.3 years) and demonstrated that infliximab monotherapy or infliximab plus AZA combination therapy is superior

  placebo group compared with more than 40 weeks in the infliximab group (P<0.001).

24

 24

45

 45

  with luminal disease should first be assessed for risk factors of disease progression. These at-risk patients may require a more intensive treatment than those without risk factors.

  AZA/6-MP throughout the 52-week trial. The primary end point was clinical remission (Crohn's Disease Activity Index [CDAI] <150) off steroids. Significantly more patients receiving

  SONIC trial on the treatment algorithm The recently published SONIC (Study of Biologic and Immunomodulator Naïve Patients in Crohn's

15 (

 15 ie, hospitalisations/surgeries), mucosal healing has become a recognised clinical end point with infliximab since it is linked to improved outcomes.

  CRP levels or mucosal lesions should, therefore, be used to identify patients who are particularly likely to benefit from an infliximab-based treatment strategy.Data from SONIC change the traditional CD treatment algorithm. It demonstrates that early infliximab-based treatment leads to improved outcomes that include rapid symptomatic improvement, sustained steroid-free remission and complete mucosal healing compared with AZA monotherapy in AZA-naïve patients. In addition, patients with evidence of active disease (high CRP/mucosal lesions) derive the most benefit from an infliximab-based treatment strategy. It should be noted that although the present discussion focuses on the impact of SONIC on the CD treatment algorithm, results from the Step-up Top-down (SUTD) study 17 may be considered for further early intervention as a next step. Briefly, the SUTD study assessed the efficacy of combined immunosuppressive therapy (AZA 2.5 mg/kg plus 3 infusions of infliximab 5 mg/kg) or conventional management (sequential treatment with corticosteroids, immunosuppressant and infliximab) in newly diagnosed, treatment-naïve patients with CD.

  benefit:risk ratio of infliximab or any other biologic needs to be considered before treating patients.

  previous loss of response to infliximab. After 4 weeks of treatment, 52% (82 of 159) of patients in this group achieved a 70-point response (decrease in baseline in CDAI score of 70 points or more)

  receiving anti-TNF therapy. It is recommended that live vaccines not be given concurrently while receiving infliximab.

  an infliximab-based treatment strategy, especially in AZA-naïve patients with high inflammatory burden at baseline, provides the most benefit in improving therapeutic outcomes.

Fistulising CD (infliximab) 12 • 12 Paediatric CD (infliximab) 12 • 12 •

 12121212 Treatment of fistulising, active CD in adult patients who have not responded despite a full and adequate course of therapy with conventional treatment (including antibiotics, drainage and immunosuppressive therapy) Treatment of severe, active CD in paediatric patients aged 6 to 17 years who have not responded to conventional therapy, including a corticosteroid, an immunomodulator and primary nutrition therapy, or who are intolerant to or have contraindications for such therapies Adult UC(infliximab) Treatment of moderately to severely active UC in adult patients who have had an inadequate response to conventional therapy, including corticosteroids and 6-mercaptopurine (6-MP) or azathioprine (AZA), or who are intolerant to or have medical contraindications for such therapies Table

Figure 1 .

 1 Figure 1. Treatment algorithm for complex fistulising disease with infliximab.

Figure 2 .Figure 3 .

 23 Figure 2. Treatment algorithm for luminal disease with risk factors. * Normal CRP, steroid-free clinical remission with mucosal healing.

31 †Figure 4 .

 314 Figure 4. Full Infliximab-based treatment algorithm. * Normal CRP, steroid-free clinical remission with mucosal healing. † Presence of risk factors may determine mono versus combination therapy.

Table 1 .

 1 Lichtenstein GR, Feagan BG, Cohen RD, et al. Serious infections and mortality in association with therapies or Crohn's disease: TREAT registry. Clin Gastroenterol Hepatol. 2006;4:621-630. European indications for infliximab and adalimumab in CD and/or UC • Treatment of severe, active CD in adult patients who have not responded despite a full and adequate course of therapy with a corticosteroid and/or an immunosuppressant, or who are intolerant to or have medical contraindications for such therapies

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Table 3 .

 3 /morbidity[START_REF] Steinhart | Induction therapy in Crohn's disease[END_REF] Other risk factors for progressive disease in luminal CD• Terminal ileal location[START_REF] Solberg | Clinical course in Crohn's disease: results of a Norwegian population-based ten-year follow-up study[END_REF]

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Arlicot Aurore
email: aurore.arlicot@polytech.univ-nantes.fr

Normand Nicolas
email: nicolas.normand@polytech.univ-nantes.fr

Guédon Jeanpierre
email: jean-pierre.guedon@polytech.univ-nantes.fr

Amouriq Yves
email: yves.amouriq@univ-nantes.fr

Extraction of Bone Structure with a Single-Scan Skeletonization Driven by Distance

Shape description is an important step in image analysis. Skeletonization methods are widely used in image analysis since they are a powerful tool to describe a shape. This paper presents a new single-scan skeletonization using different diskrete distances. The application of this method is the extraction of caracteristics from µCT images in order to estimate the bone state.

I. INTRODUCTION

A skeleton (example image 2(d)) is a one-point wide line centered in the shape which keeps the shape topology. The skeletonization process is reversible if the local width (distance to the background) is kept in the skeleton.

There exist different skeletonization algorithms. Thinning methods [START_REF] Kong | Digital topology: Introduction and survey[END_REF]- [START_REF] Lohou | A 3D 6-subiteration curve thinning algorithm based on P-simple points[END_REF] peel the shape layer by layer while preserving topology, until the skeleton is obtained. However, the result depends on the order of pixel deletions and the skeleton may not be well centered.

Distance-based method [START_REF] Attali | Du squelette discret ou continu[END_REF], [START_REF] Thiel | Unification de la squelettisation menée en distance[END_REF] identify pixels centered in the shape (centers of maimal disks, also known as medial axis points). These points are usually diskonnected and an extra step is needed to reconnect them.

The method used in this paper is based on the principle of the Distance Ordered Homotopic Thinning (DOHT [START_REF] Pudney | Distance-ordered homotopic thinning: a skeletonization algorithm for 3D digital images[END_REF]). This method peels the shape according to the distance information. Commonly, at least two scans of the image are needed for the skeleton computation in the state-of-the-art skeletonization methods. Two scans are necessary to compute the distance map and the number of thinning scans depends on the maximal width of the shape. In this work, a single scan algorithm was obtained by considering information propagation in order to compute the skeleton. Our skeletonization method is based on a thinning driven distance. That is to say we peel the shape considering the distance. The medial axis pixels appear in the pixel skeleton subset. This new method changes how information is propagated in the image by translating the centers of the disks, so that we only need one scan to compute the distance map [START_REF] Normand | Path-based distance with varying weights and neighborhood sequences[END_REF], as well as the medial axis and the skeleton.

The application of this study aims to extract the main characteristics of trabecular bones in order to characterize bone structures [START_REF] Guggenbuhl | Texture analysis of X-ray radiographs of iliac bone is correlated with bone micro-CT[END_REF], [START_REF] Arlicot | A single scan skeletonization algorithm: application to medical imaging of trabecular bone[END_REF].

In Section II the distance map translated (DT ′ ) and the medial axis translated (M A ′ ) computation will be presented. With the DT ′ and M A ′ , the computation of the skeleton will be presented in the Section III. The medical application will be presented in Section IV.

II. THE DISTANCE MAP AND MEDIAL AXIS TRANSLATED

COMPUTATION

In order to compute the skeleton, we first compute a distance map. A distance map attribute to a pixel his distance value from this pixel to the foreground.

Our method need a translation of the center of the disks to compute the distance map with one scan. So we make a translation vector -→ t so that the center of the disk is the last pixel on this disk. Figure 1 shows some translation vectors used in our algorithm. Note that with our method we change the information propagation. The distance map obtained is translated and not symmetric.

DT ′ X (p) = 0 if p / ∈ X min LU T-→ v (DT ′ x (p -- → v ))} otherwise
With the vector -→ v ∈ N where N is the set of motion vectors used in the path construction.

In a second step we compute a medial axis. Medial axis points are characterised by the center of the maximal disks.

III. THE SKELETONIZATION METHOD

After the computation of the translated distance map and the medial axis, we can read these informations in one scan of the image.

The idea of the skeletonization algorithm is to compute the real distance value during the scan of the image and in a same time compute the pixel simplicity.

Data: A binary image X, DT ′ , M A ′ Result: The skeleton S for l ← 0 to

K do for k ← 0 to L do p ← (k, l); for r ← DT ′ (p + --→ t r+1 ) to DT ′ (p + - → t r ) do if M A ′ (p) = r andp is simple then Skeleton ← 0 end end end end
There are special cases wich are described in [START_REF] Arlicot | Single-scan skeletonization driven by a neighborhood-sequence distance[END_REF]. Our algorithm is faster than the reference algorithm. Reference algorithm is a skeletonization driven by distance. Only the order of the main loops are differents than our algorithm. For an image like image 2(c) the execution time of the reference algorithm is 0.0288 seconds and the execution time of our algorithm is 0.0143 seconds.

IV. APPLICATION OF THE SKELETONIZATION

We aim at studying the bone architecture. Bone is composed of trabecula and, on radiography, we can see trabecular area. This trabecular area is studied here to analyse the bone structure.

The skeleton of the trabecular area allows for computing the quality of this bone structure. From the skeleton we are able to compute :

• the number of terminal points;

• the number of intersection points;

• the length of each trabecula;

• the width of each trabecula. In order to identify the special points we define:

• A terminal point is a special point which have only one neighbor, • A intersection point is a special point which have more (or equal) than 3 neighbors and have 3 or more component connected in his 8 neighborhoods.

V. CONCLUSION AND FURTHER WORKS

The skeletonization method presented in this paper needs one scan of the image. This method can be applied with any N S distances. The method presented in this paper is faster than the reference algorithm.

A further work is to compute the length of each trabecula and their width (according to the distance map).

Fig. 1 .

 1 Fig. 1. Translation vectors

Fig. 2 .

 2 Fig. 2. Example of trabecular bone and cortical bone.