M Pleszczyńska

A Wiater

J Szczodrak

Mutanase from Paenibacillus sp. MP-1 produced inductively by fungal -1,3-glucan and its potential for the degradation of mutan and Streptococcus mutans biofilm

Keywords: 1,3-Glucan, Laetiporus sulphureus, mutanase, Paenibacillus Microbial enzymes, Microbiology, Biotechnology

Mutanase from sp. MP-1 produced inductively by fungal α-1,3-glucan and its potential for the degradation of mutan and biofilm

M. Pleszczyńska, A. Wiater, J.

Introduction

Dental plaque is a biofilm formed by the adhesion of Streptococcus mutans and other bacterial cells to the tooth surface. This adhesion is mediated by extracellular polysaccharides (EPS), whose important components are homopolymers, such as mutans ( -1,3-glucans) produced by cariogenic streptococci. EPS are mostly waterinsoluble and have a complex structure. They promote selective adherence and accumulation of mutans streptococci on teeth and increase the bulk and porosity of dental plaque (Paes Leme et al. 2006). Formation of dental plaque leads to dental caries and other oral diseases, but enzymatic hydrolysis of mutans can prevent and/or remove dental biofilm. Degradation of -1,3-glucans can be performed by two types of enzymes, endomutanases (EC 3.2.1.59) and exomutanases (EC 3.2.1.84), which release short oligosaccharides or glucose from the polysaccharide chain, respectively. Mutanases are produced by fungi, mainly Trichoderma, and also by several bacterial genera, such as Bacillus, Paenibacillus, Flavobacterium, Streptomyces, and Pseudomonas.

Previous studies indicate mutanases could be used to prevent caries [START_REF] Asai | Mutanase-containing oral compositions[END_REF][START_REF] Inoue | Inhibition of dental plaque formation by mouthwash containing an endo-alpha-1,3 glucanase[END_REF][START_REF] Tsuchiya | Effect of recombinant mutanase and dextranase on plaque removal[END_REF]). However, their use in oral and dental care depends on the availability of their inducers.

The mutanases reported so far have been inducible in media containing streptococcal mutan. Unfortunately, this biopolymer is not available in bulk quantities due to the pathogenicity of its producers (cariogenic streptococci), the necessity to use complex and expensive media (e.g., beef brain-heart infusion), multistage production process, low product yields (no more than 2 g l -1 ), and high structural heterogeneity. Substitution of mutan by more available inducers could facilitate commercial-scale mutanase production.

An alternative source of -1,3-glucans is the fungal cell wall. We have shown that a cell wall preparation from Laetiporus sulphureus effectively induced mutanase in Trichoderma harzianum [START_REF] Wiater | Mutanase induction in Trichoderma harzianum by cell wall of Laetiporus sulphureus and its application for mutan removal from oral biofilms[END_REF]) and, to our best knowledge, this was the first occasion when this material had been used for mutanase production. We have also described a newly isolated, mutan-degrading bacterium, Paenibacillus sp. MP-1, which synthesizes high amounts of extracellular mutanase when grown on bacterial mutan [START_REF] Pleszczyńska | Paenibacillus strain MP-1: a new source of mutanase[END_REF]. In this present study, L. sulphureus cell wall preparations, together with other fungal -1,3-glucans, have been tested as inducers of Paenibacillus mutanase. The induced mutanase hydrolyzed streptococcal mutan and also had biofilm-degrading activity.

Materials and methods

Microorganisms

Paenibacillus sp. MP-1 was isolated from soil and was characterized as closely related to P. curdlanolyticus [START_REF] Pleszczyńska | Paenibacillus strain MP-1: a new source of mutanase[END_REF]

Mutan preparation

Mutan was synthesized from sucrose by cell-free glucosyltransferases of Streptococcus sobrinus/downei CCUG 21020. A form of this glucan treated by dextranase (Sigma) was also prepared and used as a mutanase assay substrate [START_REF] Pleszczyńska | Methods for obtaining active mutanase preparations from Paenibacillus curdlanolyticus[END_REF][START_REF] Wiater | Optimization of conditions for the efficient production of mutan in streptococcal cultures and post-culture liquids[END_REF]. A 1 H NMR analysis of the native and dextranase-treated glucans confirmed that they were mutans with 59.1 and 86.6 mol % -1,3-linked glucosyl residues, respectively.

Isolation of cell wall material and an alkali-soluble glucan from fruiting bodies of Laetiporus sulphureus

Fresh fruiting bodies of L. sulphureus were lyophilized and milled and the dried fruiting bodies (DFB, 100 g) were suspended in water, heated at 117 C for 1 h, and washed with water by centrifugation (9600 g, 20 min).

The material treated three times in this way was lyophilized and used as a water-insoluble cell-wall preparation (CWP, 70 g) [START_REF] Wiater | Mutanase induction in Trichoderma harzianum by cell wall of Laetiporus sulphureus and its application for mutan removal from oral biofilms[END_REF]. For the isolation of the alkali-soluble -glucan, the DFB fraction (100 g) was successively extracted with methanol, NaCl (9 g l -1 ), hot water, Na 2 CO 3 (50 g l -1 ), and 1 M NaOH containing 0.2 g NaBrH 4 l -1 for 24 h at room temperature. The final extract was neutralized with 1 M HCl under constant mixing [START_REF] Kiho | Polysaccharides in fungi. XXXIV. A polysaccharide from the fruiting bodies of Amanita muscaria and the antitumor activity of its carboxymethylated product[END_REF]. The water-insoluble fraction was washed with water, collected by centrifugation, and lyophilized (purified -1,3-glucan preparation, 56.9 g).

Preparation of mycelia of filamentous fungi

Aspergillus wentii, A. nidulans, A. niger CBS 554.65, and A. fumigatus CCM F-614 were cultured in Hasegawa medium [START_REF] Hasegawa | Enzymes that hydrolyze fungal cell wall polysaccharides. I. Purification and properties of an endo--D-(1 3)-glucanase from Trichoderma viride[END_REF]) on a rotary shaker at 150 rpm and 25 C for 6 days. Mycelia were harvested, washed with water, lyophilized, and milled.

Biofilm assay

Mutanase preparation was obtained by salting out with ammonium sulfate of cell-free filtrates obtained after culture of Paenibacillus MP-1 on a medium containing CWP. Biofilm degradation assays were performed by a modified method of [START_REF] Shimotsuura | Biochemical and molecular characterization of a novel type of mutanase from Paenibacillus sp. strain RM1: Identification of its mutan-binding domain, essential for degradation of Streptococcus mutans biofilms[END_REF]. Streptococcus sobrinus/downei CCUG 21020 (formerly S. mutans OMZ 176) was inoculated into glass tubes containing 3 ml BHI medium with 30 mg sucrose ml -1 and grown for 24 h at 37 C at an angle of 30 . The medium was then discarded and the formed biofilms were rinsed twice with 4 ml distilled water. Next, 4 ml of each mutanase solution (0.01; 0.1; 0.2; 1, and 2 U ml -1 in 50 mM phosphate buffer, pH 6.6) were added to the tubes. After 3 min at 37 C, the enzyme was replaced with 4 ml buffer, and incubation was continued for 6 h. In control trials, biofilms were incubated with the mutanase solutions for the whole 6 h. At the end of the experiments, residual biofilms were rinsed twice with 4 ml of water, homogenized, lyophilized, and weighed.

Analytical methods

To determine mutanase activity, a reaction mixture containing 0.5 ml of 0.2% (w/v) suspension of homogenized, dextranase-treated mutan in 0.05 M potassium phosphate buffer (pH 6.6) and 0.5 ml of appropriately diluted enzyme was incubated at 45 C. After 60 min the insoluble mutan was removed by centrifugation (15,500 g for 10 min), and the released reducing sugars were estimated by the Samogyi-Nelson method using D-glucose as a standard. One unit of mutanase activity (U) was defined as the amount of the enzyme which produced 1 mol reducing sugar equivalent per min under the defined conditions. The specific activity was expressed as mutanase units per mg protein. Protein was quantified using a protein assay kit (Bio-Rad) with bovine serum albumin as a standard. All the experiments and determinations were performed in triplicate, and the results were expressed as mean values. Standard deviations in the results were less than 5%.

Results

Effect of carbon sources on Paenibacillus sp. MP-1 mutanase production

Several -1,3-glucan preparations derived from selected fungi were tested as potential Paenibacillus MP-1 mutanase inducers. The culture medium was supplemented with mutan as a control; in other trials, mutan was substituted by equivalent amounts of fungal -glucan preparations. Table 1 shows that the -1,3-glucan from the fruiting bodies of Laetiporus sulphureus induced high mutanase activity. Practical application of Laetiporus -1,3-glucan, however, is conditioned by the cost and accessibility of its preparations. Among the three forms of fruiting body preparations tested as carbon sources, (a) an extensively purified -1,3-glucan, (b) a cell-wall preparation (CWP), and (c) lyophilized and milled fruiting bodies (DFB), the first one induced the highest mutanase activity (Table 2) but was eliminated from further experiments due to its high price. DFB and CWP which are relatively inexpensive forms of Laetiporus -1,3-glucan, gave similar levels of mutanase activity, but DFB contained water-soluble substances which made long-term storage difficult. The CWP fraction was storage-stable and was therefore used as a mutanase inducer. CWP concentrations for maximum enzyme production (0.35 U ml -1 ) ranged from 1.5 to 2 g l -1 .

Mutan and biofilm degradation

Fig. 1 shows that the cell-wall-induced mutanase preparation effectively degraded streptococcal mutan.

The total amount of reducing sugars in 6-hour hydrolysates was 0.23 mg ml -1 (23%) and increased to 27% after 24 h. The CWP-induced mutanase solubilized 83% of mutan within 6 h. The enzyme also effectively degraded matrix -1,3-glucans in biofilms preformed by S. mutans on a glass surface, even when the enzyme acted on them for 3 min only (Fig. 2). The rate of biofilm degradation depended on the mutanase dose. Maximum dissociation of adherent biofilm (64%) occurred at the highest enzyme concentration (2 U ml -1 ).

Discussion

Typically, mutanase synthesis is induced by -1,3-glucan. The mutan produced by cariogenic streptococci is a commonly used and effective mutanase inducer, but has no practical application in biotechnology; larger-scale production of mutanase would require the use of more available and safer inducers derived from other sources. Such inducers can be found in cell walls of a large number of fungi.

Only a few substrates containing fungal -1,3-glucan had been investigated previously for their ability to induce bacterial -1,3-glucanases. [START_REF] Meyer | Purification and properties of (1 3)--glucanases from Bacillus circulans WL-12[END_REF] obtained 0.31 U glucanase ml -1 from Bacillus circulans WL-12 supplementing the media with whole cells of Schizosaccharomyces pombe or purified -1,3-glucan from Aspergillus niger. Streptomyces KI-8 produced mutanase (0.16 U ml -1 ) when it was cultured on -1,3-glucan isolated from dried fruiting bodies of Lentinus edodes [START_REF] Imai | Properties of an -1,3-glucanase from Streptomyces sp. KI-8[END_REF]. A cell-wall preparation of Schizophyllum commune was used as a mutanase inducer in Bacillus circulans KA-304, but the enzyme activities were not reported (Mizuno et al. 1997;[START_REF] Yano | Occurrence of a specific protein in Basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304 inductively with a cellwall preparation of Schizophyllum commune[END_REF][START_REF] Yano | Cloning and expression of an -1,3-glucanase gene from Bacillus circulans KA-304: The enzyme participates in protoplast formation of Schizophyllum commune[END_REF]. Importantly, none of those substrates were rich in -1,3-glucan, and, in most cases, their preparation was expensive and time-consuming.

Furthermore, the mentioned -1,3-glucan-induced glucanases had not been considered in terms of their ability to hydrolyze dental-plaque mutan. Only Streptomyces KI-8 α-1,3-glucanase showed a weak ability to degrade this glucan [START_REF] Imai | Properties of an -1,3-glucanase from Streptomyces sp. KI-8[END_REF]. About 9 and 10% hydrolysis was achieved for two mutans of S. mutans (glucan solubilization reached 38.0 and 42.0%) after 24 h. However, to our knowledge, similarly to the other mentioned mutanases, the Streptomyces mutanase has not been applied for the degradation of oral biofilms.

Laetiporus sulphureus is a valuable source of -1,3-glucan for mutanase production. This has already been demonstrated for mutanase from Trichoderma harzianum [START_REF] Wiater | Mutanase induction in Trichoderma harzianum by cell wall of Laetiporus sulphureus and its application for mutan removal from oral biofilms[END_REF], but this -glucan has never been used as an inducer of bacterial mutanases. L. sulphureus belongs to parasitic basidiomycota infecting hardwood trees as a brown rot of the heartwood of living trees and as a saprophyte on dead trees. It grows throughout much of the world and produces very large edible shelf-shaped fruiting bodies of pink-orange color.

Fruiting bodies of L. sulphureus can contain even up to 88% of -1,3-glucan in the wall material (other fungi up to 46%). The linear -glucan predominantly consists of glucose (96%), which is mainly -1,3-linked [START_REF] Grün | Evidence for two conserved mechanisms of cell-wall -glucan biosynthesis in fungi[END_REF][START_REF] Jelsma | Observations of the cell-wall compositions of the bracket fungi Laetiporus sulphureus and Piptoporus betulinus[END_REF][START_REF] Wiater | Mutanase induction in Trichoderma harzianum by cell wall of Laetiporus sulphureus and its application for mutan removal from oral biofilms[END_REF]. Fruiting bodies and cultured mycelium of Laetiporus are readily available on a large scale.

-1,3-Glucan preparations from fruiting bodies of Laetiporus sulphureus are capable of inducing a satisfactory level of mutanase activity (0.35 U ml -1 ) in Paenibacillus sp. MP-1, which is higher than or comparable to the activities of this enzyme from other bacteria [START_REF] Imai | Properties of an -1,3-glucanase from Streptomyces sp. KI-8[END_REF][START_REF] Matsuda | Purification and properties of mutanase from Bacillus circulans[END_REF][START_REF] Meyer | Purification and properties of (1 3)--glucanases from Bacillus circulans WL-12[END_REF]. Furthermore, contrary to other fungal--1,3-glucan-induced glucanases, CWP-induced Paenibacillus mutanase is suitable for application in dentistry. It not only effectively degrades pure streptococcal mutan in a long-time reaction, but can also remove water-insoluble glucan from the polymer matrix of preformed Streptococcus biofilm, even if it is allowed to act on the biofilm for a very short time. To date, only [START_REF] Shimotsuura | Biochemical and molecular characterization of a novel type of mutanase from Paenibacillus sp. strain RM1: Identification of its mutan-binding domain, essential for degradation of Streptococcus mutans biofilms[END_REF] have reported a similar efficacy of mutanase. They obtained an almost complete degradation of S. mutans ATCC 25175 biofilm after 3-min incubation with Paenibacillus sp. RM1 mutanase at 1.4 U ml -1 .

In summary, the results of this study may have implications in dental caries prevention. The easilyavailable and inexpensive L. sulphureus cell-wall preparations can substitute streptococcal mutan in the production of high mutanase activity from Paenibacillus sp. MP-1. The obtained enzyme shows high hydrolytic activity towards mutan and quickly and effectively degrades bacterial biofilm. These characteristics suggest its potential application as an active agent of oral care compositions for controlling dental biofilm. Mutan saccharification () and mutan solubilization (). Native mutan (10 mg) and 1U of non-purified mutanase were added to 10 ml of 0.05 M phosphate buffer (pH 6.6) and incubated at 40 C together with 0.05 % NaN 3 (w/v) for 24 h. Samples were withdrawn periodically and analyzed for total reducing sugars. Solubilization of insoluble glucan was measured by turbidimetric analysis at 560 nm and the degree of solubilization was calculated. 100 % of saccharification or solubilization corresponds to the total glucose residues of the initial amount of mutan (1.1 mg ml -1 ) or the turbidity of the blank without mutan (OD 560 close to or equal to zero), respectively.

Figure captions Fig. 1

 captions1 Figure captions

Fig. 2

 2 Fig. 2 Degradation of S. mutans biofilms by Paenibacillus MP-1 mutanase induced by L. sulphureus cell wall. 6h action of mutanase (), 3-min action of mutanase (). The rate of biofilm degradation was calculated with the following equation: [1weight of mutanase-treated biofilm/ weight of untreated biofilm] 100 %. One hundred percent biofilm degradation corresponds to complete removal of mutan from biofilm matrix (weight of mutanase-treated biofilm close to zero).

  Fig. 1

  . The fruiting bodies of Laetiporus sulphureus (Bul.: Fr.) Murrill were obtained from infected trees growing in Lublin and area, Poland. A voucher specimen was deposited in the Department of Industrial Microbiology, Maria Curie-Skłodowska University, Poland. Aspergillus wentii and A. nidulans were taken from the culture collection of that department. Dried fruiting bodies of Lentinus edodes, Pleurotus djamor,

	P. ostreatus, P. citrinopileatus, P. eryngii, Agaricus blazei, Kuehneromyces mutabilis, Hericium erinaceum, and
	Piptoporus betulinus were a kind gift from Prof. M. Siwulski (Department of Vegetable Crops, Poznań
	University of Life Sciences, Poland).

Table 1

 1

Table 1

 1 Inductive effect of -1,3-glucan preparations of different origins on the production of extracellular

	mutanase by Paenibacillus MP-1 a
	-1,3-Glucan sources b	Mutanase activity
		(U ml -1 )	(U mg -1 protein)
	Streptococcal mutan	0.36	1.57
	Fruiting bodies of:
	Laetiporus sulphureus	0.21	1.06
	Pleurotus djamor	0.04	0.38
	Pleurotus ostreatus	0.03	0.22
	Pleurotus citrinopileatus	0.02	0.17
	Lentinus edodes	0.01	0.11
	Mycelium of:
	Aspergillus wentii	0.17	0.62
	Aspergillus niger	0.07	0.27
	a The MP-1 strain was grown in a modified Kanzawa et al. (1994) medium composed of (in g l -1 ): carbon source
	-lyophilized and milled inducer, 5 (with the exception of mutan and fruiting bodies of L. sulphureus, 2.5);
	Bacto-tryptone (Difco), 10; (NH 4 ) 2 HPO 4 , 1.5; KH 2 PO 4 , 1; MgSO 4 7H 2 O, 0.5; CaCl 2 2H 2 O, 0.5; NaCl, 0.01;
	MnCl 2 4H 2 O, 0.01, and FeCl 3 6H 2 O, 0.01; pH 6.8. The medium (75 ml in 500-ml flasks) was inoculated with 5
	% (v/v) Paenibacillus preculture (24-hours old) and incubated on a rotary shaker at 170 rpm and 30 C. Enzyme
	activity was determined at the time of maximum mutanase production (48 96 h).

b Pleurotus erynii, Agaricus blazei, Kuehneromyces mutabilis, Hericium erinaceum, Piptoporus betulinus, Aspergillus fumigatus, and Aspergillus nidulans were also used as -1,3-glucan sources, but gave negative results.

Table 2

 2 Inductive effect of -1,3-glucan preparations isolated from fruiting bodies of Laetiporus sulphureus on

	the production of extracellular mutanase by Paenibacillus sp. MP-1 a
	-1,3-glucan preparations from Laetiporus sulphureus		Mutanase activity
		U ml -1	U mg -1 protein
	Purified -1,3-glucan (2.5 g l -1 )	0.66	1.95
	Dried fruiting bodies (2.5 g l -1 )	0.33	1.46
	Cell wall preparation (g l -1 )
	1.0	0.22	1.02
	1.5	0.33	1.18
	2.0	0.35	1.17
	2.5	0.27	1.02
	3.0	0.04	0.19

a Kanzawa medium modified by addition of 10 g tryptone l -1 , pH 6.8; 170 rpm; 30 C; 48 h

Acknowledgments

This work was financially supported from funds for science in the years 2008 -2011 as the development project No 325/R/P01/2007/IT1.

The authors declare that they have no conflict of interest.


Javier P Gisbert
email: gisbert@meditex.es

María Chaparro

Maria Esteve

Javier ; Gisbert

Javier P Gisbert

María Esteve

M.D. Javier P Gisbert

Prevention and management of hepatitis B and C infection in patients with inflammatory bowel disease

Keywords: Inflammatory bowel disease < Disease-based, Immunosuppression < Topics, Large intestine < Organ-based, Viral hepatitis Crohn's disease, ulcerative colitis, hepatitis B virus, hepatitis C virus, inflammatory bowel disease, interferon, tumor necrosis factor, vaccine

Introduction

Viral hepatitis is a very common infection: over 350 million people worldwide have chronic hepatitis B virus (HBV) infection and over 250 million people have chronic hepatitis C virus (HCV) infection [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF] .

In the last ten years, the treatment of inflammatory bowel disease (IBD), which includes both Crohn's disease (CD) and ulcerative colitis (UC), has been marked by the increasing use of immunosuppressors (mainly azathioprine/mercaptopurine and methotrexate) and by the advent of biological therapies. Increasing evidence in favor of immunosuppressors means that they are being used more often and earlier in the course of the disease.

Many issues concerning the relationship between IBD and HBV/HCV infection remain unresolved. For example, the prevalence of HBV and HCV infection in IBD patients has received little attention and it is still unclear which patients should be screened for HBV/HCV and when. Such questions are particularly relevant, as immunomodulator therapy has a clear impact on the natural history of viral hepatitis, although it is not clear which factors increase the risk of HBV reactivation in IBD patients treated with immunosuppressants. Deciding on the most suitable candidates for antiviral prophylaxis and timing of this treatment are also matters of debate, as is the type of prophylaxis to be chosen. Although a consensus exists on the need to vaccinate IBD patients against HBV infection, vaccination seems to be underused in clinical practice. Furthermore, the efficacy of HBV vaccination, specifically in IBD patients, and the factors influencing this efficacy, are unknown. Several types, doses, and schedules of HBV vaccination have been recommended in the general population, and it has been suggested that modified dosing regimens might increase response rates in IBD patients. Whether testing for serological immunity should be systematically performed after HBV vaccination in patients with IBD remains controversial, as does the most suitable cut-off titer for antibodies to hepatitis B surface antigen (anti-HBs). It has been suggested that a second course of HBV vaccination is recommendable in IBD patients whose first vaccination attempt fails, but the effectiveness of this rescue strategy has not been proven. We do not know how long anti-HBs antibody titers remain positive after HBV vaccination and, consequently, whether anti-HBs titers should be periodically monitored and whether booster vaccines should be considered. Finally, issues concerning the efficacy and safety of antiviral treatment in patients with IBD have not been resolved, and more information is required on the risk of exacerbation of IBD in patients treated with interferon.

The present manuscript poses a serious of questions on the relationship between IBD and HBV/HCV infection and attempts to answer them using scientific evidence in order to provide practical conclusions for the clinician who diagnoses and treats patients with IBD.

Bibliographical searches were performed in MEDLINE up to September 2010 using the following key words (all fields): ("inflammatory bowel disease" OR "Crohn's disease" OR "ulcerative colitis") AND hepatitis.

What is the prevalence of HBV and HCV infection in IBD patients?

Knowledge of the prevalence of HBV and HCV infection in patients with IBD is relevant, because the virus can reactivate under immunosuppressive therapy (see below). However, little information is available on the prevalence of HBV and HCV infection in patients with IBD. In the late 1990s, patients with IBD were considered to be at risk of HCV and HBV infection, probably due to prior surgery or blood transfusions [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF][START_REF] Biancone | Hepatitis B and C virus infection in Crohn's disease[END_REF][START_REF] Forns | Nosocomial transmission of HCV in the liver unit of a tertiary care center[END_REF][START_REF] Salcedo-Mora | Chronic hepatitis C and Crohn's disease: nosocomial infection treatment with PEG-interferon plus ribavirin[END_REF][START_REF] Broome | Liver disease in ulcerative colitis: an epidemiological and follow up study in the county of Stockholm[END_REF] . a) Prevalence of HBV infection: Some relatively "old" studies have reported a significantly higher prevalence of HBV infection in IBD patients than in controls. For example, Biancone et al [START_REF] Biancone | Hepatitis B and C virus infection in Crohn's disease[END_REF] reported that 11% of IBD patients had antibodies to hepatitis B core protein (anti-HBc), a statistically significantly higher figure than in controls. This high prevalence was related to blood transfusions and surgical procedures, suggesting nosocomial transmission of the virus. In contrast, other studies have reported HBV exposure rates in IBD patients that were similar to those of the IBD patients consecutively recruited at 17 Spanish hospitals [START_REF] Loras | Prevalence and factors related to hepatitis B and C in inflammatory bowel disease patients in Spain: a nationwide, multicenter study[END_REF] , found the prevalence of HBV infection to be lower than that reported in previous studies (less than 1% hepatitis B surface antigen (HBsAg)-positive and less than 10% anti-HBc-positive). However, no differences were found between CD and UC, in contrast with other studies, where the prevalence of HBV markers was higher in CD than in UC [START_REF] Biancone | Hepatitis B and C virus infection in Crohn's disease[END_REF] .

Studies evaluating the prevalence of HBV infection in patients with IBD are summarized in , 6-9 . The weighted mean prevalence of HBsAg-positive infection in the 5 studies included (total population, 3,121 patients) was 1.03%. The corresponding mean value for anti-HBc-positive infection was 8.1%. When only patients with CD were considered, the mean values for HBsAg and anti-HBc were 1.04% and 7.2%, while for UC patients the values were 0.82% and 8.1%.

b) Prevalence of HCV infection:

Over the past decade, patients with IBD have been considered at risk for HBV/HCV infection, perhaps due to nosocomial transmission. However, large series such those reported by Biancone et al in Italy revealed that the prevalence of previous HCV infection (demonstrated by the presence of anti-HCV antibodies) was not significantly higher in CD patients than in controls (7.4% versus 5.1%), and that it was actually lower in UC patients than in controls [START_REF] Biancone | Hepatitis B and C virus infection in Crohn's disease[END_REF] . REPENTINA 1 revealed an anti-HCV-positive rate of 2.3% in CD patients and 1.3% in UC patients [START_REF] Loras | Prevalence and factors related to hepatitis B and C in inflammatory bowel disease patients in Spain: a nationwide, multicenter study[END_REF] . Therefore, as with HBV infection, prevalence of HCV infection in patients with IBD was concluded to be similar to that of the general reference population and lower than that in previously published series. This fact, in addition to the lack of association with invasive procedures, suggests the existence of adequate preventive measures in centers attending these patients [START_REF] Loras | Prevalence and factors related to hepatitis B and C in inflammatory bowel disease patients in Spain: a nationwide, multicenter study[END_REF] .

Table 2 presents a series of studies evaluating the prevalence of HCV infection in patients with IBD [START_REF] Biancone | Hepatitis B and C virus infection in Crohn's disease[END_REF][START_REF] Loras | Prevalence and factors related to hepatitis B and C in inflammatory bowel disease patients in Spain: a nationwide, multicenter study[END_REF][START_REF] Esteve | Chronic hepatitis B reactivation following infliximab therapy in Crohn's disease patients: need for primary prophylaxis[END_REF][START_REF] Chevaux | Prevalence of hepatitis B and C and risk factors for nonvaccination in inflammatory bowel disease patients in Northeast France[END_REF][START_REF] Longo | Prevalence of hepatitis C in patients with chronic inflammatory bowel disease in the region of Nice and evaluation of risk factors[END_REF][START_REF] Bargiggia | Is interferon-alpha therapy safe and effective for patients with chronic hepatitis C and inflammatory bowel disease? A case-control study[END_REF] . The weighted mean prevalence of anti-HCV-positive infection for the 6 studies (3,575 patients) was 3.25%. When only patients diagnosed with CD were considered, the mean value for anti-HCV was 3.4%, while the respective figure for UC patients was 2.7%. In summary, more than one decade ago, patients with IBD were considered to be at risk of HBV and HCV infection. Blood transfusion and surgery were thought to be the routes, thus raising the hypothesis of nosocomial transmission. However, recent epidemiologic surveys have found that prevalence in IBD patients is similar to or even lower than in the general population. At present, therefore, patients with IBD do not appear to be at risk of hepatitis, probably as a result of safety measures for blood transfusions, improved surgical asepsis, and universal anti-HBV vaccination.

Which IBD patients should be screened for prior HBV/HCV exposure, and when?

Traditionally, the results of viral (mainly HBV) serology testing has been thought necessary before patients with IBD can start immunosuppressive therapy [START_REF] Viget | Opportunistic infections in patients with inflammatory bowel disease: prevention and diagnosis[END_REF] . Today, it is generally accepted that all IBD patients should be screened for exposure to HBV. In fact, screening for HBV should be performed at diagnosis of IBD, rather than waiting until initiation of therapy with immunomodulators or tumor necrosis factor-α (TNF) antagonists [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF][START_REF] Melmed | Patients with inflammatory bowel disease are at risk for vaccine-preventable illnesses[END_REF][START_REF] Melmed | Vaccination strategies for patients with inflammatory bowel disease on immunomodulators and biologics[END_REF][START_REF] Wasan | A practical guide to vaccinating the inflammatory bowel disease patient[END_REF] . This recommendation is based on the potential fatal consequences of HBV reactivation and the availability of safe and effective anti-HBV drugs to prevent reactivation 1 . However, no consensus has been reached for HCV screening prior to starting immunomodulators [START_REF] Rahier | European evidence-based Consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease[END_REF] . As the risk of HCV reactivation under immunosuppressive drugs appears to be very low or non-existent (see below), systematic screening of IBD patients cannot be definitively recommended. Reactivation of HBV is an important concern for patients taking immunosuppressants and can manifest in many ways, from a subtle change in serum aminotransferase levels to fulminant hepatic failure and death [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF] . Reactivation of HBV infection (with liver dysfunction, including fulminant hepatitis) is a well-described complication of immunosuppression in the setting of organ transplantation or cancer chemotherapy, occurring in up to 50% of patients where concomitant antiviral therapy is not used [START_REF] Mindikoglu | Hepatitis B virus reactivation after cytotoxic chemotherapy: the disease and its prevention[END_REF][START_REF] Lok | Chronic hepatitis B[END_REF][START_REF] Firpi | Management of viral hepatitis in hematologic malignancies[END_REF] . Mortality from fulminant liver failure after reactivation of HBV in patients receiving chemotherapy is reported in 4-60% of cases [START_REF] Kohrt | Antiviral prophylaxis for chemotherapy-induced reactivation of chronic hepatitis B virus infection[END_REF] . In this section, the effect of immunosuppressants (corticosteroids, thiopurines, and anti-TNF drugs) on HBV infection is reviewed.

What is the impact of immunosuppressive therapy on the natural history of HBV and HCV infection? Which factors increase the risk of HBV reactivation in patients with IBD treated with immunosuppressants? a) Effect of immunosuppressants on HBV infection

Corticosteroids: Fatal viral reactivation has been described mainly in the context of malignant hematological disease among patients receiving chemotherapy combined with corticosteroids [START_REF] Chevaux | Inflammatory bowel disease and hepatitis B and C[END_REF] . In patients with lymphoma, reactivation of HBV replication is more common when chemotherapy regimens include corticosteroids [START_REF] Cheng | Steroid-free chemotherapy decreases risk of hepatitis B virus (HBV) reactivation in HBV-carriers with lymphoma[END_REF] . In one report, HBV reactivation in a patient with UC treated with prednisone (plus azathioprine) resulted in fulminant hepatic failure requiring liver transplantation [START_REF] Zeitz | Hepatic failure due to hepatitis B reactivation in a patient with ulcerative colitis treated with prednisone[END_REF] .

Thiopurines: A recent study described two cases of hepatic flare (hepatic decompensation with ascites in one case) thought to be caused by treatment of acute CD with systemic corticosteroids and/or azathioprine [START_REF] Sacco | Entecavir for hepatitis B virus flare treatment in patients with Crohn's disease[END_REF] .

Anti-TNF drugs: Reactivation of HBV infection related to infliximab has been reported in a number of patients with diseases other than IBD [START_REF] Yeo | Frequency of hepatitis B virus reactivation in cancer patients undergoing cytotoxic chemotherapy: a prospective study of 626 patients with identification of risk factors[END_REF][START_REF] Ostuni | Hepatitis B reactivation in a chronic hepatitis B surface antigen carrier with rheumatoid arthritis treated with infliximab and low dose methotrexate[END_REF][START_REF] Michel | Fulminant hepatitis after infliximab in a patient with hepatitis B virus treated for an adult onset still's disease[END_REF][START_REF] Yeo | Hepatitis B virus reactivation in breast cancer patients receiving cytotoxic chemotherapy: a prospective study[END_REF][START_REF] Oniankitan | Infliximab therapy for rheumatic diseases in patients with chronic hepatitis B or C[END_REF][START_REF] Wendling | Reactivation of a latent precore mutant hepatitis B virus related chronic hepatitis during infliximab treatment for severe spondyloarthropathy[END_REF] . Table 3 presents a summary of studies reporting reactivation of HBV after infliximab, specifically in patients with IBD (CD in all cases) [START_REF] Esteve | Chronic hepatitis B reactivation following infliximab therapy in Crohn's disease patients: need for primary prophylaxis[END_REF][START_REF] Del Valle Garcia-Sanchez | Infliximab therapy in a patient with Crohn's disease and chronic hepatitis B virus infection[END_REF][START_REF] Ueno | Infliximab therapy for Crohn's disease in a patient with chronic hepatitis B[END_REF][START_REF] Millonig | Subfulminant hepatitis B after infliximab in Crohn's disease: need for HBV-screening?[END_REF][START_REF] Colbert | Fulminant hepatic failure in chronic hepatitis B on withdrawal of corticosteroids, azathioprine and infliximab for Crohn's disease[END_REF][START_REF] Esteve | Lamivudine resistance and exacerbation of hepatitis B in infliximab-treated Crohn's disease patient[END_REF][START_REF] Loras | Liver dysfunction related to hepatitis B and C in patients with inflammatory bowel disease treated with immunosuppressive therapy[END_REF][START_REF] Madonia | Occult hepatitis B and infliximab-induced HBV reactivation[END_REF][START_REF] Ojiro | Reactivation of hepatitis B in a patient with Crohn's disease treated using infliximab[END_REF] .

Cases of reactivation after treatment with infliximab have been described in occult carriers of HBV who are negative for HBsAg and positive for anti-HBc antibody [START_REF] Madonia | Occult hepatitis B and infliximab-induced HBV reactivation[END_REF] (see occult HBV infection, below). Reactivation of HBV infection in HBsAg carriers occurred as soon as after the first infliximab infusion and as late as 2 years after starting infliximab (Table 3), indicating that this Almost all cases of HBV reactivation associated with infliximab have occurred in patients receiving concomitant treatment with other immunosuppressors, such as corticosteroids or thiopurines (Table 3), suggesting that more profound immunosuppression may facilitate viral reactivation. Lamivudine was the main agent administered and, although clinical and analytical recovery was achieved in most of the cases, three patients died. There are no case reports of reactivation of HBV with adalimumab or certolizumab pegol; however, as these are newer TNF antagonists, the risk of reactivation would be expected to be a class effect [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF] .

Reactivation of HBV infection may be severe if prophylaxis is not administered [START_REF] Esteve | Lamivudine resistance and exacerbation of hepatitis B in infliximab-treated Crohn's disease patient[END_REF] . Some years ago, Esteve et al [START_REF] Esteve | Chronic hepatitis B reactivation following infliximab therapy in Crohn's disease patients: need for primary prophylaxis[END_REF] reported three CD patients with chronic hepatitis B infection treated with infliximab. Two patients experienced reactivation: the infection resolved in one patient and the other patient died. The third patient, who received concomitant lamivudine, experienced no variations in hepatic clinical or biochemical parameters. In a recent review, Shale et al [START_REF] Shale | Review article: chronic viral infection in the anti-tumour necrosis factor therapy era in inflammatory bowel disease[END_REF] found reports of anti-TNF drugs administered to HBV patients (with and without IBD); outcomes ranged from apparent viral clearance to fatal hepatitis. Most of the patients treated with anti-TNF drugs who did not receive concomitant antiviral therapy had increased viral loads and serum aminotransferase, and many developed clinically apparent hepatic dysfunction [START_REF] Shale | Review article: chronic viral infection in the anti-tumour necrosis factor therapy era in inflammatory bowel disease[END_REF] .

In previous studies, treatment with a combination of immunosuppressants was identified as the most consistent factor related to the risk of de novo and/or reactivation of opportunistic infections in IBD [START_REF] Toruner | Risk factors for opportunistic infections in patients with inflammatory bowel disease[END_REF][START_REF] Garcia-Vidal | Risk factors for opportunistic infections in infliximab-treated patients: the importance of screening in prevention[END_REF] . The largest study assessing outcome of HBV infection in patients with IBD and its relation to immunosuppressive therapy has recently been published (REPENTINA 2) [START_REF] Loras | Liver dysfunction related to hepatitis B and C in patients with inflammatory bowel disease treated with immunosuppressive therapy[END_REF] . The study sample comprised patients with IBD and viral hepatitis from 19 Spanish hospitals. Liver dysfunction was observed in 9 of 25 (36%) HBsAg-positive patients, 6 of whom developed hepatic failure. Treatment with ≥ 2 immunosuppressants was an independent predictor of reactivation. In other words, most patients without reactivation received only one immunosuppressant for a short period and/or antiviral prophylaxis [START_REF] Loras | Liver dysfunction related to hepatitis B and C in patients with inflammatory bowel disease treated with immunosuppressive therapy[END_REF] . REPENTINA 2 shows that no single immunosuppressant seems to be more involved in the development of liver dysfunction, which is probably a consequence of combined immunosuppression. In addition, it also demonstrates that the risk of reactivation under immunosuppressive treatment is clearly higher than spontaneous reactivation as part of the natural history of the infection. Thus, the risk of reactivation of HBV seems to be related to the magnitude of immunosuppression.

In summary, HBV carriers with IBD who require immunosuppressive therapy (e.g., corticosteroids, methotrexate or azathioprine) or biological agents (e.g., anti-TNF agents) frequently present reactivation of HBV infection. This reactivation can be severe and may even prove fatal.

b) Effect of immunosuppressants on HCV infection

Corticosteroids: It seems reasonable to assume that corticosteroids used to treat IBD have no detrimental effect on the course of HCV [START_REF] Rahier | European evidence-based Consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease[END_REF] . However, one patient with CD had an acute flare of HCV infection on discontinuing corticosteroids [START_REF] Biancone | Immunomodulatory drugs in Crohn's disease patients with hepatitis B or C virus infection[END_REF] . Furthermore, available data on recurrence in the graft after liver transplantation suggest that corticosteroids should be tapered slowly to limit the risk of early recurrent HCV hepatitis and the risk of cholestatic fibrosing hepatitis [START_REF] Magy | Effects of corticosteroids on HCV infection[END_REF][START_REF] Vivarelli | Influence of steroids on HCV recurrence after liver transplantation: A prospective study[END_REF] .

Thiopurines/methotrexate: In vitro, azathioprine has antiviral activity against HCV [START_REF] Stangl | Effect of antimetabolite immunosuppressants on Flaviviridae, including hepatitis C virus[END_REF] . Data from patients undergoing liver transplantation for HCV infection indicate that azathioprine can be used in HCV-infected patients with IBD [START_REF] Rahier | European evidence-based Consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease[END_REF] . A study of a small series of HCV-infected patients with arthropathy showed no detrimental effect of treatment with methotrexate [START_REF] Nissen | Rheumatological manifestations of hepatitis C: incidence in a rheumatology and non-rheumatology setting and the effect of methotrexate and interferon[END_REF] .

Anti-TNF drugs: Case series suggest that anti-TNF therapy has no adverse effects and that it might even improve HCV infection [START_REF] Oniankitan | Infliximab therapy for rheumatic diseases in patients with chronic hepatitis B or C[END_REF][START_REF] Campbell | Infliximab therapy for Crohn's disease in the presence of chronic hepatitis C infection[END_REF][START_REF] Holtmann | Treatment of patients with Crohn's disease and concomitant chronic hepatitis C with a chimeric monoclonal antibody to TNF[END_REF][START_REF] Magliocco | Etanercept therapy for patients with psoriatic arthritis and concurrent hepatitis C virus infection: report of 3 cases[END_REF][START_REF] Parke | Anti-tumor necrosis factor agents for rheumatoid arthritis in the setting of chronic hepatitis C infection[END_REF][START_REF] Zein | Etanercept as an adjuvant to interferon and ribavirin in treatment-naive patients with chronic hepatitis C virus infection: a phase 2 randomized, double-blind, placebo-controlled study[END_REF][START_REF] Vauloup | Effects of tumor necrosis factor antagonist treatment on hepatitis C-related immunological abnormalities[END_REF][START_REF] Roux | Safety of anti-TNF-alpha therapy in rheumatoid arthritis and spondylarthropathies with concurrent B or C chronic hepatitis[END_REF][START_REF] Aslanidis | Inhibition of TNFalpha does not induce viral reactivation in patients with chronic hepatitis C infection: two cases[END_REF][START_REF] Pitarch | Treatment of psoriasis with adalimumab[END_REF][START_REF] Abdelmalek | Successful treatment of chronic hepatitis C with pegylated interferon, ribavirin, and infliximab in a patient with Crohn's disease[END_REF] . Some authors [START_REF] Peterson | Effect of tumour necrosis factor alpha antagonists on serum transaminases and viraemia in patients with rheumatoid arthritis and chronic hepatitis C infection[END_REF] found no significant differences in liver function or viremia at baseline or during follow-up in HCV-infected patients receiving infliximab or etanercept for rheumatoid arthritis. Finally, etanercept improved virological response to combined [START_REF] Zein | Etanercept as an adjuvant to interferon and ribavirin in treatment-naive patients with chronic hepatitis C virus infection: a phase 2 randomized, double-blind, placebo-controlled study[END_REF] . Etanercept is not used to treat IBD, but its apparent lack of side effects in HCV suggests that other TNF-antagonists may also be low-risk [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF][START_REF] Shale | Review article: chronic viral infection in the anti-tumour necrosis factor therapy era in inflammatory bowel disease[END_REF] .

The largest series assessing the outcome of HCV infection in patients with IBD and its relation to immunosuppressive therapy revealed liver dysfunction in 8 of 51 HCV-infected patients (16%) [START_REF] Loras | Liver dysfunction related to hepatitis B and C in patients with inflammatory bowel disease treated with immunosuppressive therapy[END_REF] .

Most were related to corticosteroids alone and in one case to azathioprine. However, except for one case with simultaneous positive antibodies against HIV (AIDS) and anti-HBc (suggesting occult HBV infection), liver dysfunction related to HCV was very mild.

In summary, the use of immunomodulators (e.g., corticosteroids, azathioprine or methotrexate) in IBD patients with HCV appears to be low-risk. The magnitude of the reactivation is clearly lower for HCV than for HBV [START_REF] Loras | Liver dysfunction related to hepatitis B and C in patients with inflammatory bowel disease treated with immunosuppressive therapy[END_REF][START_REF] Vento | Reactivation of replication of hepatitis B and C viruses after immunosuppressive therapy: an unresolved issue[END_REF] . At present, there are no data to suggest that TNF-antagonists are unsafe in IBD patients with HCV.

How frequent is progression to liver cirrhosis in IBD patients receiving immunosuppressants?

The proportion of IBD patients with liver cirrhosis and the mean time to development of cirrhosis in both HBV-infected and HCV-infected patients is as reported in the non-IBD population, considering the natural history of both infections [START_REF] Loras | Liver dysfunction related to hepatitis B and C in patients with inflammatory bowel disease treated with immunosuppressive therapy[END_REF] . In this sense, progression to liver cirrhosis seems to be similar to that previously observed in non-immunosuppressed infected patients [START_REF] Seeff | The history of the "natural history" of hepatitis C (1968-2009)[END_REF][START_REF] Mcmahon | The natural history of chronic hepatitis B virus infection[END_REF] .

Thus, administration of immunosuppressants, as is common in IBD, does not seem to increase progression to end-stage liver disease. This contrasts with observations in HCV-infected patients who progress to liver cirrhosis (and even hepatocarcinoma) after liver transplantation [START_REF] Iacob | Predictors of graft and patient survival in hepatitis C virus (HCV) recipients: model to predict HCV cirrhosis after liver transplantation[END_REF][START_REF] Scherzer | Efficacy and safety of antiviral therapy in patients with Crohn's disease and chronic hepatitis C[END_REF][START_REF] Beckebaum | Predictive factors of outcome in patients transplanted for hepatitis B[END_REF] .

Who should receive antiviral prophylaxis and when?

Although data for IBD patients are scarce, prophylaxis has proven beneficial in patients undergoing chemotherapy. Thus, randomized controlled trials have shown the benefit of prophylaxis with lamivudine to prevent reactivation of HBV in this group [START_REF] Jang | A randomized controlled study of preemptive lamivudine in patients receiving transarterial chemo-lipiodolization[END_REF][START_REF] Lau | Early is superior to deferred preemptive lamivudine therapy for hepatitis B patients undergoing chemotherapy[END_REF][START_REF] Hsu | A revisit of prophylactic lamivudine for chemotherapy-associated hepatitis B reactivation in non-Hodgkin's lymphoma: a randomized trial[END_REF] . One meta-analysis of lamivudine for prophylaxis of HBV reactivation in immunosuppressed patients included 21 studies and showed that this drug reduced mortality [START_REF] Katz | Lamivudine prevents reactivation of hepatitis B and reduces mortality in immunosuppressed patients: systematic review and meta-analysis[END_REF] .

The American Association for the Study of Liver Disease (AASLD) [START_REF] Association | EASL Clinical Practice Guidelines: management of chronic hepatitis B[END_REF] , the European Association for the Study of the Liver (EASL) [START_REF] Association | EASL Clinical Practice Guidelines: management of chronic hepatitis B[END_REF] , and the European Crohn and Colitis Organization (ECCO) [START_REF] Rahier | European evidence-based Consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease[END_REF] recommend early introduction of nucleoside/nucleotide analogues for all HBsAg-positive patients requiring immunosuppressive therapy. Antiviral therapy should be administered to those patients who exhibit positive HBsAg titers with or without active viral replication. Prophylaxis should be prescribed irrespective of the number and type of immunosuppressants administered, whether corticosteroids, immunomodulators, or biologics [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF] .

HBV prophylaxis is currently recommended in HBsAg-positive patients 7 days prior to initiation of chemotherapy. It should be maintained for 6 months to 1 year after completion of chemotherapy, as HBV reactivation may occur after chemotherapy is discontinued de Franchis, 2003 #804] 1, 18, 69 .

In IBD patients in particular, it is recommended to start antiviral prophylaxis 1-3 weeks prior to the introduction of immunosuppressive therapy and continue it for 6 months after withdrawal [START_REF] Esteve | Chronic hepatitis B reactivation following infliximab therapy in Crohn's disease patients: need for primary prophylaxis[END_REF][START_REF] Rahier | European evidence-based Consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease[END_REF][START_REF] Roux | Safety of anti-TNF-alpha therapy in rheumatoid arthritis and spondylarthropathies with concurrent B or C chronic hepatitis[END_REF] .

What is the relevance of occult HBV infection? Should antiviral prophylaxis be administered?

Occult HBV infection is defined as persistence of B virus genomes in HBsAg-negative individuals [START_REF] Raimondo | Statements from the Taormina expert meeting on occult hepatitis B virus infection[END_REF] . This condition is more frequent (almost 10% in some series [START_REF] Loras | Prevalence and factors related to hepatitis B and C in inflammatory bowel disease patients in Spain: a nationwide, multicenter study[END_REF] ) in patients with HBV markers (anti-HBc with or without anti-HBs). Thus, although AASLD guidelines recommend screening for HBsAg and anti-HBs, screening for anti-HBc has also been recommended to detect occult HBV [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF][START_REF] Chevaux | Inflammatory bowel disease and hepatitis B and C[END_REF] .

Before prescribing immunosuppressive therapy, the physician must take into account that IBD patients may have a reactivation of occult HBV infection. Reactivation has been repeatedly observed in patients with hematological malignancies treated with chemotherapy [START_REF] Iannitto | Hepatitis B virus reactivation and alemtuzumab therapy[END_REF][START_REF] Law | Fatal reactivation of hepatitis B post-chemotherapy for lymphoma in a hepatitis B surface antigen-negative, hepatitis B core antibody-positive patient: potential implications for future prophylaxis recommendations[END_REF] . Consequently, anti-HBc-positive patients should be treated with caution, because they are potential carriers of occult HBV [START_REF] Iannitto | Hepatitis B virus reactivation and alemtuzumab therapy[END_REF][START_REF] Law | Fatal reactivation of hepatitis B post-chemotherapy for lymphoma in a hepatitis B surface antigen-negative, hepatitis B core antibody-positive patient: potential implications for future prophylaxis recommendations[END_REF] .

In a Spanish multicenter trial, liver dysfunction was observed in 36% of HBsAg-positive patients [START_REF] Loras | Liver dysfunction related to hepatitis B and C in patients with inflammatory bowel disease treated with immunosuppressive therapy[END_REF] . On the contrary, no definite HBV reactivations have been found in anti-HBc-positive patients lacking HBsAg [START_REF] Chevaux | Prevalence of hepatitis B and C and risk factors for nonvaccination in inflammatory bowel disease patients in Northeast France[END_REF][START_REF] Loras | Liver dysfunction related to hepatitis B and C in patients with inflammatory bowel disease treated with immunosuppressive therapy[END_REF] . There is only one report of HBV reactivation in an anti-HBcpositive/HBsAg-negative patient with CD treated with corticosteroids and infliximab [START_REF] Madonia | Occult hepatitis B and infliximab-induced HBV reactivation[END_REF] . Therefore, systematic use of antiviral prophylaxis in anti-HBc-positive patients lacking HBsAg cannot be recommended [START_REF] Yeo | Frequency of hepatitis B virus reactivation in cancer patients undergoing cytotoxic chemotherapy: a prospective study of 626 patients with identification of risk factors[END_REF][START_REF] Loras | Liver dysfunction related to hepatitis B and C in patients with inflammatory bowel disease treated with immunosuppressive therapy[END_REF][START_REF] Lok | Reactivation of hepatitis B virus replication in patients receiving cytotoxic therapy. Report of a prospective study[END_REF] . This approach differs from that recommended for patients undergoing chemotherapy, particularly with rituximab. In these patients, antiviral prophylaxis is recommended if anti-HBc is detected-even in patients lacking HBsAg-based on reports of severe HBV reactivation in this setting [START_REF] Yeo | Hepatitis B virus reactivation in lymphoma patients with prior resolved hepatitis B undergoing anticancer therapy with or without rituximab[END_REF] . In any case, liver function and HBV DNA should be periodically monitored in anti-HBc-positive patients with or without anti-HBs during immunosuppressive therapy, since occult HBV carriers are more frequent in this group [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF][START_REF] Chevaux | Inflammatory bowel disease and hepatitis B and C[END_REF] .

Which antiviral prophylaxis should be chosen for IBD patients?

In chronic HBsAg-positive carriers, antiviral prophylaxis with nucleotide/nucleoside analogues is recommended before administering immunosuppressive agents [START_REF] Rahier | European evidence-based Consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease[END_REF] . According to specific guidelines for HBV treatment [START_REF] Rahier | European evidence-based Consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease[END_REF][START_REF] Lok | Chronic hepatitis B[END_REF] , patients with high baseline HBV DNA levels (>2,000 IU/ml) should continue antiviral treatment until endpoints applicable to non-immunosuppressed patients are reached.

If immunosuppressive therapy is expected to last more than 1 year, nucleotide/nucleoside analogues with a lower propensity than lamivudine for generating drug-resistant mutations of HBV DNA might be preferred [START_REF] Rahier | European evidence-based Consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease[END_REF] . Lamivudine resistance commonly develops with prolonged use, and has been detected in up to 30% of patients after 1 year and 70% by 5 years. The emergence of resistance has also been associated with reactivation in patients on long-term anti-TNF therapy [START_REF] Esteve | Lamivudine resistance and exacerbation of hepatitis B in infliximab-treated Crohn's disease patient[END_REF][START_REF] Liaw | Hepatitis flares and hepatitis B e antigen seroconversion: implication in anti-hepatitis B virus therapy[END_REF][START_REF] Conjeevaram | Management of chronic hepatitis B[END_REF] [START_REF] Esteve | Chronic hepatitis B reactivation following infliximab therapy in Crohn's disease patients: need for primary prophylaxis[END_REF] reported a case of resistance in a patient with CD who had been taking lamivudine prophylaxis for 5 years. This agent may be appropriate for a short course of prophylaxis during chemotherapy, but immunosuppressive medications for IBD may be required indefinitely. Some CD patients, mainly those with a fistulizing pattern, require long-term therapy with biologics to maintain remission. Thus, alternative antiviral medications for HBV, such as tenofovir or entecavir, are preferred in these cases, as they have the lowest rates of resistance with long-term use [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF][START_REF] Viget | Opportunistic infections in patients with inflammatory bowel disease: prevention and diagnosis[END_REF][START_REF] Lok | Chronic hepatitis B: update 2009[END_REF] . In this setting, entecavir is preferred to adefovir because of its rapid action and lack of nephrotoxicity.

Nevertheless, there has recently been a report of an HBV flare with entecavir in patients with concurrent CD [START_REF] Sacco | Entecavir for hepatitis B virus flare treatment in patients with Crohn's disease[END_REF] . Interferon-α is best avoided for 2 reasons: first, it can exacerbate CD and, second, it can cause additional bone marrow suppression [START_REF] Rahier | European evidence-based Consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease[END_REF] .

Who should be vaccinated against HBV infection and when?

Universal immunization beginning at birth and other successful HBV vaccination strategies have resulted in a dramatic reduction in HBV transmission in many countries with historically high endemicity [START_REF]Hepatitis B vaccines: WHO position paper--recommendations[END_REF] . In patients diagnosed with IBD, the present method for prevention of opportunistic infections rests on vaccinations and on a thorough clinical and laboratory work-up before administration of immunosuppressors and/or biologics [START_REF] Viget | Opportunistic infections in patients with inflammatory bowel disease: prevention and diagnosis[END_REF] .

Patients with IBD are very likely to need immunosuppressive therapy in the course of their disease. Furthermore, response rates to some vaccines, for example HBV vaccine, are considerably lower in patients receiving immunosuppressive agents or anti-TNF therapy (see below) [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF][START_REF] Sands | Guidelines for immunizations in patients with inflammatory bowel disease[END_REF] .

Therefore, given the recommendation to vaccinate before starting treatment with immunosuppressive agents, the best time for immunization is at diagnosis of IBD [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF][START_REF] Viget | Opportunistic infections in patients with inflammatory bowel disease: prevention and diagnosis[END_REF][START_REF] Melmed | Patients with inflammatory bowel disease are at risk for vaccine-preventable illnesses[END_REF][START_REF] Melmed | Vaccination strategies for patients with inflammatory bowel disease on immunomodulators and biologics[END_REF][START_REF] Wasan | A practical guide to vaccinating the inflammatory bowel disease patient[END_REF] . In spite of the aforementioned recommendations and the presence of significant risk factors in IBD patients [START_REF] Viget | Opportunistic infections in patients with inflammatory bowel disease: prevention and diagnosis[END_REF] , vaccines are underprescribed, indicating that immunization against selected vaccinepreventable illnesses-including viral hepatitis-is uncommon. Melmed et al [START_REF] Melmed | Patients with inflammatory bowel disease are at risk for vaccine-preventable illnesses[END_REF] assessed risk of exposure and immunization status among patients receiving care in an IBD specialty clinic. Patients completed a self-administered, pretested, structured questionnaire during a routine visit for the management of IBD. Their responses indicated that, although 44% of the study population had at least 1 risk factor for hepatitis B (e.g., blood transfusion or tattoos), only 28% had been vaccinated against HBV infection. In another study, 49% of gastroenterologists had never recommended vaccinations for IBD patients [START_REF] Gupta | Vaccination And Screening For Infections In Patients With Inflammatory Bowel Disease: A Survey Of Australian Gastroenterologists[END_REF] . Finally, a Spanish cross-sectional multicenter study [START_REF] Loras | Prevalence and factors related to hepatitis B and C in inflammatory bowel disease patients in Spain: a nationwide, multicenter study[END_REF] detected vaccination against HBV in only 12% of IBD patients.

Are IBD patients correctly vaccinated against HBV infection in clinical practice?

The most commonly cited reason for non-immunization in these patients is "never being offered the vaccine" or "didn't know I needed it" [START_REF] Sands | Guidelines for immunizations in patients with inflammatory bowel disease[END_REF] . Consequently, it is necessary to increase awareness of immunization guidelines among health care providers and their patients [START_REF] Sands | Guidelines for immunizations in patients with inflammatory bowel disease[END_REF] .

In summary, the low rates of vaccinationputs IBD patients at risk of infections (e.g., HBV infection) that might easily be avoided through a more rigorous and standardized vaccination program [START_REF] Wasan | A practical guide to vaccinating the inflammatory bowel disease patient[END_REF] . Such an approach could limit the infectious complications associated with immunomodulating and biologic therapies, and should be a priority for all gastroenterologists [START_REF] Wasan | A practical guide to vaccinating the inflammatory bowel disease patient[END_REF] .

Fortunately, implementation of universal HBV vaccination programs in developed countries will minimize this risk in the future, as confirmed by the results of vaccination programs in younger patients.

How effective is HBV vaccination?

The primary 3-dose vaccine series induces protective antibody concentrations in >95% of healthy infants, children, and young adults [START_REF]Hepatitis B vaccines: WHO position paper--recommendations[END_REF][START_REF] Coates | Hepatitis B vaccines: assessment of the seroprotective efficacy of two recombinant DNA vaccines[END_REF][START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF] revealed that seroprotection (> 10 mIU/ml of anti-HBs titers) was achieved in 96% and 94% of patients, respectively, using the 3-dose schedule (0, 1, and 6 months).

However, immunosuppressive illnesses in general are associated with reduced immunogenicity following vaccination [START_REF]Hepatitis B vaccines: WHO position paper--recommendations[END_REF] . Published data suggest that patients with diseases other than CD and UC who received vaccinations while on immunosuppressive therapy may have a suboptimal serologic response with waning protective antibody titers after a variety of vaccinations [START_REF] Melmed | Vaccination strategies for patients with inflammatory bowel disease on immunomodulators and biologics[END_REF] . Such a response has been observed in non-IBD patients treated with long-term immunosuppressive agents [START_REF] Bruguera | Impaired response to recombinant hepatitis B vaccine in HIV-infected persons[END_REF][START_REF] Elkayam | Safety and efficacy of vaccination against hepatitis B in patients with rheumatoid arthritis[END_REF] .

The rate of response to vaccines in IBD patients has received little attention. IBD patients who received a booster immunization with tetanus and diphtheria toxoids produced inadequate levels of antibody titers after vaccination [START_REF] Stevens | Defective generation of tetanusspecific antibody-producing B cells after in vivo immunization of Crohn's disease and ulcerative colitis patients[END_REF] . The response rate to HBV vaccine seems to be quite low, mainly in patients receiving immunosuppressive agents or anti-TNF therapy [START_REF] Sands | Guidelines for immunizations in patients with inflammatory bowel disease[END_REF] . For example, in the study by Melmed et al, only 33% (3/9) of the subgroup of patients who were immunized had detectable anti-HBs antibody titers [START_REF] Melmed | Patients with inflammatory bowel disease are at risk for vaccine-preventable illnesses[END_REF] . In the study by Vida et al [START_REF] Perez | Adequate rate of response to hepatitis B virus vaccination in patients with inflammatory bowel disease[END_REF] , where 43% of IBD patients had received immunosuppressive medication before the first vaccine dose, only 36% had adequate levels of anti-HBs antibodies (i.e., > 10 mIU/ml). Finally, Chaparro et al [START_REF] Chaparro | Immune response to hepatitis B vaccination in patients with inflammatory bowel disease[END_REF] assessed the efficacy of HBV vaccine (double dosage at 0, 1, and 2 months) in 211 patients with IBD, and found that only 60% of patients had adequate anti-HBs antibody levels (defined as > 10 mIU/ml). If the cut-off point for adequate immunity was set at > 100 mIU/ml (as some guidelines recommend; see below), the efficacy rate was as low as 34%. In summary, the response rate to HBV vaccine seems to be quite low in IBD patients.

What factors affect the efficacy of HBV vaccination in IBD patients?

In diseases other than IBD, the efficacy of HBV vaccination is affected by the type and number of immunomodulators given [START_REF] Wagner | Failure of vaccination against hepatitis B with Gen H-B-Vax-D in immunosuppressed heart transplant recipients[END_REF][START_REF] Worns | Incidence of HAV and HBV infections and vaccination rates in patients with autoimmune liver diseases[END_REF] . For example, some studies have suggested that patients on TNF inhibitors may have a slightly decreased immune response to influenza vaccine [START_REF] Mamula | Immune response to influenza vaccine in pediatric patients with inflammatory bowel disease[END_REF][START_REF] Lu | Immune response to influenza vaccine in children with inflammatory bowel disease[END_REF][START_REF] Gelinck | The effect of anti-tumour necrosis factor alpha treatment on the antibody response to influenza vaccination[END_REF] . Specific data for patients with IBD are very scarce. Some authors have evaluated the serologic response to influenza vaccine in patients with IBD and observed that those receiving anti-TNF therapy were less likely to be seroprotected [START_REF] Lu | Immune response to influenza vaccine in children with inflammatory bowel disease[END_REF] . Others have demonstrated a suboptimal response to pneumococcal vaccination in patients with IBD who are receiving combination therapy with immunosuppressants plus anti-TNF drugs [START_REF] Melmed | Immunosuppression impairs response to pneumococcal polysaccharide vaccination in patients with inflammatory bowel disease[END_REF] .

In healthy individuals, several risk factors for non-response have been identified, including immunosuppressive disease, smoking, older age, male sex, and high body mass index [START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF] . Only two studies have provided data on the factors affecting efficacy of HBV vaccine in IBD patients. Vida et al [START_REF] Perez | Adequate rate of response to hepatitis B virus vaccination in patients with inflammatory bowel disease[END_REF] assessed the immune status of 129 IBD patients receiving HBV vaccine (at 0, 1, and 6 months), and the only factor which correlated with failure of the anti-HBs response to the vaccine was older age. However, concomitant use of immunomodulators or biologics did not affect the efficacy of HBV vaccination. The small sample size (only ten patients were treated with biologics) and the corresponding low statistical power of this study may explain the lack of statistically significant differences. In fact, a tendency toward a lower rate of response to HBV vaccine was found in patients under immunosuppressive treatment (mainly azathioprine). In the study by Chaparro et al [START_REF] Chaparro | Immune response to hepatitis B vaccination in patients with inflammatory bowel disease[END_REF] , overall, 34% of patients had adequate anti-HBs antibody levels (defined as > 100 mIU/ml); however, the response rate was much lower in patients on anti-TNF therapy (10% vs. 40%), although this difference did not reach statistical significance, again probably due to the small sample size. Patients with rheumatoid arthritis treated with adalimumab can be immunized effectively with pneumococcal and influenza vaccines [START_REF] Kaine | Immune responses following administration of influenza and pneumococcal vaccines to patients with rheumatoid arthritis receiving adalimumab[END_REF] .

The lack of response to HBV vaccine by IBD patients may be a consequence of associated immunosuppressive/biologic treatment rather than of IBD per se. For example, the rate of response to HBV vaccine has been reported to be lower in patients with celiac disease than in healthy individuals [START_REF] Ahishali | Response to hepatitis B vaccination in patients with celiac disease[END_REF] . However, the response to HBV vaccine in celiac children who were compliant with a gluten-free diet is no different from that of the healthy population [START_REF] Ertem | The response to hepatitis B vaccine: does it differ in celiac disease?[END_REF] . Thus, celiac disease may be one of the immune diseases associated with a reduced response to HBV vaccine; however, this lack of response might not be permanent, and a gluten-free diet and compliance with treatment may ameliorate the immune response to the vaccine [START_REF] Ertem | The response to hepatitis B vaccine: does it differ in celiac disease?[END_REF] . In this respect, Melmed et al [START_REF] Melmed | Immunosuppression impairs response to pneumococcal polysaccharide vaccination in patients with inflammatory bowel disease[END_REF] evaluated immunogenicity with the 23-valent pneumococcal polysaccharide vaccine in IBD patients receiving both anti-TNF agents and immunomodulators, IBD patients not receiving any immunosuppressive therapy, and in healthy controls. IBD patients who received combination immunosuppressive therapy mounted a less effective immune response than patients with IBD who received nonimmunosuppressive therapy and controls, both of which exhibited a similar-good-response.

Overall, the response of IBD patients to HBV vaccine seems to be quite low, even in those patients not receiving immunosuppressive/biologic treatment [START_REF] Perez | Adequate rate of response to hepatitis B virus vaccination in patients with inflammatory bowel disease[END_REF][START_REF] Chaparro | Immune response to hepatitis B vaccination in patients with inflammatory bowel disease[END_REF] , suggesting that IBD per se may be partially responsible for the suboptimal serologic response to HBV vaccine.

Which type, dose, and schedule of HBV vaccination are advisable?

The standard vaccination schedule involves the administration of 3 doses, usually at 0, 1, and 6 months [START_REF] Viget | Opportunistic infections in patients with inflammatory bowel disease: prevention and diagnosis[END_REF][START_REF]Hepatitis B vaccines: WHO position paper--recommendations[END_REF][START_REF] Sands | Guidelines for immunizations in patients with inflammatory bowel disease[END_REF] . The second dose should be administered 1 month after the first dose; the third dose should be administered at least 2 months after the second dose (and at least 4 months after the first dose) [START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF][START_REF]Recommended adult immunization schedule: United States[END_REF] . Longer intervals may increase final anti-HBs titers, but not seroconversion rates [START_REF]Hepatitis B vaccines: WHO position paper--recommendations[END_REF][START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF] .

Accelerated immunization schedules (for example, at 0, 1, and 2 months) are effective, although anti-HBs titers are lower when intervals between injections are reduced [START_REF] Shouval | Hepatitis B vaccines[END_REF] .

If the combined hepatitis A and hepatitis B vaccine (Twinrix®) is used, 3 doses at 0, 1, and 6 months are administered. Modified dosing regimens, including doubling the standard antigen dose, might increase response rates [START_REF] Rahier | European evidence-based Consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease[END_REF][START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF][START_REF] Choudhury | Responses to hepatitis B vaccine boosters in human immunodeficiency virus-infected children[END_REF][START_REF] Fonseca | Randomized trial of recombinant hepatitis B vaccine in HIV-infected adult patients comparing a standard dose to a double dose[END_REF][100] . For example, immunocompromised patients have received Recombivax HB ® (40 µg/mL) administered in a 3-dose schedule or Engerix-B ® (2 x 20 µg/mL) administered in a 4-dose schedule (at 0, 1, 2, and 6 months) [START_REF]Recommended adult immunization schedule: United States[END_REF] It is unknown whether the duration of response of vaccine-related immunity is altered in patients with IBD (with or without immunosuppression); therefore, booster doses or modified dosing schedules would be necessary. In this respect, a 4-dose schedule (days 0, 7, and 21-30 followed by a booster dose at month 12) could be appropriate [START_REF]Recommended adult immunization schedule: United States[END_REF] .

Should testing for serological immunity be performed after HBV vaccination?

Although routine serology testing for immunity is not necessary after vaccination of adults, postvaccination testing is recommended for high-risk individuals whose subsequent clinical management depends on knowledge of their immune status, as is the case in immunocompromised persons, including IBD patients [START_REF] Lok | Chronic hepatitis B[END_REF][START_REF]Hepatitis B vaccines: WHO position paper--recommendations[END_REF][START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF] . As the response rate to HBV vaccination in IBD patients is quite low, testing for serological immunity should be performed 1-2 months after administration of the last dose of the vaccine series to determine the need for revaccination [START_REF]Hepatitis B vaccines: WHO position paper--recommendations[END_REF][START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF] .

Which is the best cut-off point (anti-HBs titers) for defining adequate immunity after HBV vaccination?

Assessment of the level of vaccine-induced anti-HBs that protects against HBV infection has revealed that all patients who attained a peak antibody response to vaccination of > 10 mIU/ml were protected against carriage of HBsAg 102 . According to the World Health Organization, an anti-HBs concentration of ≥ 10 mIU/ml measured 1-3 months after administration of the last dose of the primary vaccination series is considered a reliable marker of protection against infection 9, 77, 80-81, 97, 102-104 . As time passes, anti-HBs titers frequently become undetectable. A number of long-term studies performed in different epidemiological contexts have confirmed that clinical HBV infection rarely occurs among successfully vaccinated individuals even though the anti-HBs concentrations decline to < 10 mIU/ml over time. However, among immunocompromised patients who respond to the vaccine, clinically significant HBV infection has been documented in those who do not maintain anti-HBs concentrations of ≥ 10 mIU/ml. As the persistence of detectable anti-HBs after vaccination depends on the concentration of post-vaccination antibodies, some authors have suggested that the target anti-HBs concentration might be higher for these patients. Based on this evidence, in the United Kingdom, seroprotection against HBV infection was recently redefined at ≥ 100 mIU/ml [START_REF]Hepatitis B vaccines: WHO position paper--recommendations[END_REF] .

Is a second course of HBV vaccination advisable in IBD patients in whom a first vaccination attempt fails?

Of persons who did not respond to a primary 3-dose vaccine series, 25-50% will respond to an additional dose, and 44-100% will respond to a second 3-dose course [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF][START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF] . Therefore, those patients found to have anti-HBs concentrations < 10 mIU/ml after the primary vaccine series should be revaccinated with 3 additional doses [START_REF]Hepatitis B vaccines: WHO position paper--recommendations[END_REF][START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF]105 . Furthermore, increased vaccine doses (e.g., double the standard dose) were shown to enhance revaccination response rates in one study 106 (althouh not in another 107 ). Therefore, it may be suggested to either revaccinate with the standard hepatitis B vaccine regimen using twice the standard dose or administer the combined hepatitis A and B vaccine (Twinrix) 108 . However, although among healthy individuals who did not respond to a primary 3-dose series with anti-HBs concentrations of > 10 mIU/ml, almost all respond to a 3-dose revaccination series 109 , this may not be the case in patients with IBD. Thus, in the study by Chaparro et al [START_REF] Chaparro | Immune response to hepatitis B vaccination in patients with inflammatory bowel disease[END_REF] , non-responders to the first 3-dose vaccination received a second course of vaccine (with the same 3-dose schedule), and the response (> 100 mIU/ml anti-HBs titers) to this second vaccination was only 41%. Even if patients initially form adequate antibody titers after vaccination, these titers may wane and leave patients unknowingly susceptible to infection [110][111] . Vaccine-induced anti-HBs titers decline rapidly in the first year and more gradually afterwards [START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF] . Among young adults who respond to a primary vaccine series with anti-HBs concentrations of ≥ 10 mIU/ml, up to 50% have low or undetectable concentrations (reflecting anti-HBs loss) 10-15 years after vaccination. Even when anti-HBs concentrations decline to < 10 mIU/ml, nearly all healthy persons remain protected against HBV infection [START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF] . Thus, the vaccine-induced immunologic memory is maintained for at least 12 years in non-immunocompromised persons despite the decline in anti-HBs titers 104 .

For how long do anti-HBs antibody titers remain positive after HBV vaccination? Should periodic controls (e.g., annually) be performed and booster doses considered?

Limited data are available on the duration of immune memory after hepatitis B vaccination in immunocompromised patients. In studies of long-term protection among HIV-infected persons, breakthrough infections occurring after a decline in anti-HBs concentrations to < 10 mIU/ml have been transient and asymptomatic [START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF] . However, among hemodialysis patients who responded to the vaccine, clinically significant HBV infection has been documented in those who did not maintain anti-HBs concentrations > 10 mIU/ml 112 . Therefore, immunocompromised patients could need annual testing to assess anti-HBs concentrations [START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF] . For example, for hemodialysis patients, the need for booster doses should be assessed by annual anti-HBs testing. A booster dose should be administered when anti-HBs levels decline to < 10 mIU/ml [START_REF] Mast | A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part II: immunization of adults[END_REF] . For other immunocompromised patients, the need for booster doses has not been determined. Thus, annual testing to assess anti-HBs concentrations (and administering booster doses when anti-HBs levels decline to < 10 mIU/ml) should be considered for IBD patients (perhaps only in those under immunosuppressive treatment), although more studies are necessary to establish this recommendation.

What is the efficacy of antiviral treatment for viral hepatitis in IBD patients?

Some authors have demonstrated that IBD patients with HCV infection who receive non- 21 pegylated interferon-α monotherapy have a sustained virological response that is similar to that of non-IBD controls [START_REF] Bargiggia | Is interferon-alpha therapy safe and effective for patients with chronic hepatitis C and inflammatory bowel disease? A case-control study[END_REF] . The current first-line treatment regimen for HCV infection is peginterferon-α plus ribavirin. Other authors have analyzed the efficacy of this combination regimen in CD patients with chronic hepatitis C and have concluded that efficacy is comparable to that reported in patients without CD [START_REF] Hou | Viral hepatitis and inflammatory bowel disease[END_REF][START_REF] Bargiggia | Is interferon-alpha therapy safe and effective for patients with chronic hepatitis C and inflammatory bowel disease? A case-control study[END_REF][START_REF] Scherzer | Efficacy and safety of antiviral therapy in patients with Crohn's disease and chronic hepatitis C[END_REF] .

What is the safety profile of antiviral treatment in IBD patients?

No differences in incidence of adverse events have been observed between IBD patients and non-IBD controls treated with interferon-α for HCV infection [START_REF] Bargiggia | Is interferon-alpha therapy safe and effective for patients with chronic hepatitis C and inflammatory bowel disease? A case-control study[END_REF]113 . However, Peyrin-Biroulet et al 114 reported eight cases of severe pancytopenia in patients receiving azathioprine for IBD and ribavirin plus peginterferon-α for chronic hepatitis C. The authors prospectively monitored azathioprine metabolites in two of these patients and found a dramatic increase in methylated metabolite levels. Similarly, Chaparro et al 115 reported a CD patient initially treated with azathioprine and subsequently receiving peginterferon plus ribavirin who presented with pancytopenia. These results suggest that ribavirin can interact with the metabolism of azathioprine, thus increasing the risk of developing myelotoxicity. Taking into account these observations, treatment with ribavirin and purine analogues should be individualized and patients closely monitored.

What is the risk of exacerbation of IBD with interferon?

CD is usually characterized by a Th1-type immune response. CD flares have been reported in patients taking interferon-α, which is known to stimulate a Th1-type immune response 116 .

Therefore, this agent is generally not recommended for patients with chronic HBV infection and concomitant CD, because it may exacerbate CD [START_REF] Rahier | European evidence-based Consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease[END_REF][117][118] . Nevertheless, this recommendation remains controversial, as other authors could not confirm the negative effect of interferon-α in patients with CD [START_REF] Biancone | Hepatitis B and C virus infection in Crohn's disease[END_REF][START_REF] Salcedo-Mora | Chronic hepatitis C and Crohn's disease: nosocomial infection treatment with PEG-interferon plus ribavirin[END_REF][START_REF] Holtmann | Treatment of patients with Crohn's disease and concomitant chronic hepatitis C with a chimeric monoclonal antibody to TNF[END_REF][START_REF] Abdelmalek | Successful treatment of chronic hepatitis C with pegylated interferon, ribavirin, and infliximab in a patient with Crohn's disease[END_REF] . Scherzer et al [START_REF] Scherzer | Efficacy and safety of antiviral therapy in patients with Crohn's disease and chronic hepatitis C[END_REF] analyzed the tolerability of interferon-α plus ribavirin in patients with chronic HCV infection and CD, and concluded that gastrointestinal symptoms may be temporarily exacerbated (6 of the 11 CD patients experienced exacerbation of CD-related symptoms). Nevertheless, increased CD activity is easily managed with short-term medical (glucocorticoid) therapy. Therefore, antiviral therapy with interferon-α plus ribavirin should be given to HCV-infected CD patients to prevent long-term sequelae of chronic hepatitis [START_REF] Scherzer | Efficacy and safety of antiviral therapy in patients with Crohn's disease and chronic hepatitis C[END_REF] .

Several case reports suggest a potential benefit of interferon-α in patients with UC [119][120][121][122] . However, a Cochrane review including three prospective studies of interferon-α in patients with UC concluded that interferon-α was not an effective option for this condition 123 . Patients with UC and concomitant HBV or HCV infection could receive interferon-α, since the onset of adverse effects is less likely during the course of UC than during that of CD [START_REF] Bargiggia | Is interferon-alpha therapy safe and effective for patients with chronic hepatitis C and inflammatory bowel disease? A case-control study[END_REF]113,[124][125] . However, case reports have observed new-onset UC during interferon-α treatment or exacerbation of existing UC (or IBD with a predominance of UC) 116,[124][125][126][127][128][129][130][131][132][133] . In one of these case reports, the administration of sulfasalazine allowed the patient to receive interferon-α again without a flare-up of UC 126 . This case suggests the possible efficacy of sulfasalazine therapy in patients with UC complicated by administration of interferon-α.

The review by Horn et al 134 on the treatment of HCV in the setting of IBD allows us to conclude that interferon-α does not appear to worsen the course of established IBD in patients already on maintenance therapy. The idea that HCV can be safely treated in patients with IBD in a suitable clinical context is reinforced by a retrospective study and a case-control study addressing the subject [START_REF] Bargiggia | Is interferon-alpha therapy safe and effective for patients with chronic hepatitis C and inflammatory bowel disease? A case-control study[END_REF]127 . Cottone et al retrospectively studied seven UC and seven CD patients who had undergone treatment with interferon-α monotherapy for hepatitis C, or B for at least six months 127 .

All of the patients except two had inactive IBD at the beginning of treatment, and only one of these patients had a mild relapse during therapy. The case-control study by Bargiggia et al [START_REF] Bargiggia | Is interferon-alpha therapy safe and effective for patients with chronic hepatitis C and inflammatory bowel disease? A case-control study[END_REF] In summary, interferon-α is generally safe for patients with chronic hepatitis C and IBD, provided that the latter is in clinical remission. In other words, treatment of hepatitis C does not appear to worsen the course of IBD when the disease is controlled and maintenance therapy instituted.

Conclusions

1) Recent studies demonstrate that the prevalence of both HBV infection and HCV infection in IBD patients is now similar to that of the general population. IBD patients should no longer be considered a risk group for viral hepatitis.

2) All IBD patients should be screened for HBV markers (HBsAg, anti-HBs, anti-HBc) to rule out HBV infection. The best time for screening is at diagnosis.

3) Liver dysfunction in IBD patients treated with immunosuppressants is more frequent and severe in HBV carriers than in HCV carriers and is associated with combined immunosuppression.

4)

In patients receiving anti-TNF drugs, HBV reactivation is common unless antiviral prophylaxis is administered. HBV reactivations may cause fulminant hepatic failure in patients on immunosuppressive therapy. 11) The response rate to HBV vaccination in IBD patients seems to be quite low, mainly in those already receiving anti-TNF therapy.

12) Modified HBV vaccine dosing regimens, including doubling the standard antigen dose, could increase response rates.

13) The serological response to HBV vaccine should be measured after the completion of vaccination in IBD patients.

14) An anti-HBs concentration of ≥ 10 mIU/ml measured 1-3 months after administration of the last dose of the vaccination is considered a reliable marker of protection against HBV infection; the advantages of increasing this cut-off to ≥ 100 mIU/ml are still unclear.

15) Patients with an inadequate response to the vaccination should receive a second full series (three doses) of HBV vaccine, as a relevant proportion of these patients will finally respond. 16) Periodic (e.g., annual) testing to assess anti-HBs concentrations (and administering booster doses when anti-HBs levels decline to < 10 mIU/ml) may be considered for IBD patients (perhaps only in those under immunosuppressive treatment). 17) If a patient with IBD requires treatment for HCV, the combination of peginterferon plus

  population. REPENTINA 1, a recent large-scale cross-sectional multicenter study of 2,076

  at any time during therapy with anti-TNF drugs; consequently, systematic clinical and analytical monitoring should be performed indefinitely in patients treated with these drugs.

  plus ribavirin in HCV-positive patients

  . One recently developed vaccine, which contains a new adjuvant combination (AS04) and was designed for use in patients with renal insufficiency, has been shown to have an improved immunogenicity profile 101 .

23 CD

 23 also suggests that interferon-α therapy is safe in patients with chronic hepatitis C and IBD. The authors treated ten patients and 11 UC patients with interferon-α monotherapy. All of the IBD patients were in clinical remission (or had only mildly active disease) prior to initiating interferon-α. No patient had significant worsening of CD activity index during the interferon-α treatment period or during the following 12 months.

5 ) 7 )

 57 Irrespective of the number and type of immunosuppressants administered, all HBsAgpositive patients should receive antiviral prophylaxis before starting immunosuppressant therapy in order to avoid hepatitis B flare.6) The systematic use of antiviral prophylaxis in patients with possible occult HBV -negative but anti-HBc-positive markers) does not seem to be necessary. As interferon therapy might worsen underlying IBD, nucleoside/nucleotide analogues are preferred for antiviral prophylaxis in patients with HBV. 8) Tenofovir and entecavir are preferred to lamivudine as antiviral prophylaxis of HBV, since they have lower rates of resistance in the long term. 9) As patients with IBD are very likely to need immunosuppressive therapy in the course of their disease, they should be vaccinated against HBV at diagnosis. 10) HBV vaccination is underused in IBD patients in clinical practice. The low percentage of effective vaccinations makes it advisable to intensify HBV vaccination in IBD.

  be as effective as in non-IBD patients.18)No difference in adverse events has been observed between IBD and non-IBD patients treated with interferon for HCV infection. On the other hand, ribavirin may interact with the metabolism of azathioprine, thus increasing the risk of myelotoxicity.19) Interferon is generally safe in patients with chronic hepatitis C and IBD, provided that the inflammatory bowel condition is in clinical remission.

Table

  [START_REF] Biancone | Hepatitis B and C virus infection in Crohn's disease[END_REF]

  . Esteve

	et al
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	13

  . A recent review of 181 clinical studies in which 24,277 individuals were immunized with Engerix-B and 8,627 with RECOMBIVAX HBVax II[START_REF] Coates | Hepatitis B vaccines: assessment of the seroprotective efficacy of two recombinant DNA vaccines[END_REF]

Table 1 .

 1 Studies evaluating the prevalence of hepatitis B virus infection in patients with inflammatory bowel disease (IBD).CD, Crohn's disease; UC, ulcerative colitis; HBsAg, hepatitis B surface antigen; anti-HBc, antibodies to hepatitis B core protein.

	Author	Publication	IBD type	No. of	HBsAg (%)	Anti-HBc
		year		patients		(%)
	Biancone et al 2	2001	CD	332	2.1	11
	Biancone et al 2	2001	UC	162	0.64	11.5
	Esteve et al 7	2004	CD	80	3.7	7.5
	Tolentino et al 8	2008	IBD	176	2.3	17
	Loras et al 6	2009	CD	1128	0.6	7.1
	Loras et al 6	2009	UC	928	0.8	8
	Chevaux et al 9	2010	CD	252	0.79	2.8
	Chevaux et al 9	2010	UC	63	1.59	1.6

Table 2 .

 2 Studies evaluating the prevalence of hepatitis C virus (HCV) infection in patients with inflammatory bowel disease (IBD).

	Author	Publication	IBD type	No. of	Anti-HCV
		year		patients	(%)
	Longo et al 10	2000	IBD	117	6
	Biancone et al 2	2001	CD	332	7.4
	Biancone et al 2	2001	UC	162	0.6
	Esteve et al 7	2004	CD	80	1.2
	Bargiggia et al 11	2005	CD	302	6.2
	Bargiggia et al 11	2005	UC	211	10.9
	Loras et al 6	2009	CD	1,128	2.3
	Loras et al 6	2009	UC	928	1.3
	Chevaux et al 9	2010	CD	252	0.79
	Chevaux et al 9	2010	UC	63	1.59
	CD, Crohn's disease; UC, ulcerative colitis.

Table 3 .

 3 Hepatitis B reactivations after anti-tumor necrosis factor (infliximab) in patients with Crohn's disease. The patient was previously receiving lamivudine, which was combined with adefovir dipivoxil.

	Author	Year of	HBsAg	Duration of	Immunomodulator Lamivudine Outcome
		publication	status	infliximab
				treatment
	De Valle et al 31	2004	+	6 infusions	None	Yes	Recovered
	Esteve et al 7	2004	+ ¶	4 infusions	Azathioprine	No	Recovered
	Esteve et al 7	2004	+ ¶	3 infusions	Azathioprine	Yes	Died
	Ueno et al 32	2005	+	1 infusion	None	No	Recovered
	Millonig et al 33	2006	+ ¶	4 infusions	Azathioprine	Yes	Recovered
	Colbert et al 34	2007	+	24 months	Corticosteroids	Yes	Died
					Azathioprine
	Esteve et al 35	2007	+	24 months	Azathioprine	Yes*	Recovered
	Loras et al 36	2010	+	-	Azathioprine	Yes*	Recovered
	Loras et al 36	2010	+	-	Azathioprine	Yes	Died
	Loras et al 36	2010	+	-	Corticosteroids	No	Recovered
					Azathioprine
	Madonia et al 37	2007	+ #	> 12 months	Corticosteroids	Yes	Recovered
				(6 infusions)
	Ojiro et al 38	2008	+	4 infusions	Mercaptopurine	Yes	Recovered
	HBsAg: hepatitis B surface antigen.

¶

Retrospectively found to be HBsAg-positive. # Pre-treatment HBsAg was negative, but became positive. *

Alimentary Pharmacology & Therapeutic