Detection of Helicobacter species in chronic liver disease and chronic inflammatory bowel disease

Abstract

Aim. To study the association between helicobacters and chronic liver diseases and chronic inflammatory bowel diseases.

Patients and methods. Thirty‐two patients with various chronic liver diseases and 137 patients with inflammatory bowel disease were enrolled. Antibodies to H. pylori, H. hepaticus, H. bilis, and H. pullorum were measured by enzyme immunoassay (EIA), and sera positive in a non‐pylori helicobacter EIA were further examined by immunoblot assay. Detection of Helicobacter DNA in liver biopsies was done by denaturating gradient gel electrophoresis of PCR products (PCR‐DGGE) and DNA sequence analysis.

Results. Six inflammatory bowel disease patients, four with ulcerative colitis and two with Crohn's disease, and one liver disease patient with autoimmune cholangitis had antibodies to non‐pylori helicobacters by an immunoblot assay. Four immunoblot assay‐negative patients, three with autoimmune and one with non‐autoimmune liver disease, had Helicobacter DNA in liver biopsies; three of the polymerase chain reaction (PCR) products were closely related to non‐pylori helicobacters.

Conclusion. Evidence for non‐pylori helicobacters was scant in Finnish patients with inflammatory bowel disease or chronic but not end stage liver disease. We cannot, however, rule out their role in these diseases.

• Helicobacter
• inflammatory bowel disease
• liver disease
• PCR
• Western blotting

Abbreviations
CAH	=	CHRONIC AUTOIMMUNE HEPATITIS
CD	=	Crohn's disease
EIA	=	enzyme immunoassay
IB	=	immunoblot assay
IBD	=	inflammatory bowel disease
PBC	=	primary biliary cirrhosis
PCR	=	polymerase chain reaction
PCR‐DGGE	=	denaturating gradient gel electrophoresis of PCR products
PSC	=	primary sclerosing cholangitis
UC	=	ulcerative colitis

Introduction

Helicobacter pylori (H. pylori) causes lifelong gastritis in humans. Since its discovery, 30 other formally named Helicobacter species (List of Prokaryotic Names with Standing in Nomenclature (http://www.bacterio.cict.fr) accessed 18th May 2007) have been detected, some of which have been associated with hepatitis and colitis in animals 1,2. The chronic nature of H. pylori infection has prompted increasing interest in the possible association between the non‐pylori helicobacters and chronic bowel and hepatobiliary diseases in humans. On the other hand, data suggesting that H. pylori‐induced autoantibodies may play a crucial role in the pathogenesis of autoimmune type gastric atrophy 3 have led to an idea that other Helicobacter species might have a role in the etiology of autoimmune diseases 4.

The reports on the association between helicobacters and hepatobiliary diseases in humans have revealed contradictory results 5. In most of these studies Helicobacter DNA has been detected by polymerase chain reaction (PCR) which may not always be specific 6. There are, however, two reports on the culture of a Helicobacter isolate from liver 7,8 and several reports on the demonstration of helicobacters by immunohistochemistry in human liver tissue 9–11. The growth of H. pylori is known to be inhibited by bile salts in vitro (this may not be so in vivo in the case of biliary pathology) but some enterohepatic Helicobacter species, such as H. hepaticus, H. bilis, H. pullorum, are bile‐resistant 5.

In primary biliary cirrhosis (PBC) over 95% of patients have anti‐mitochondrial antibodies that are reactive with a sub‐unit of the pyruvate dehydrogenase complex, which is considered to be the major autoantigen in PBC 12,13. Several yeasts and bacteria express an epitope similar to that found in the pyruvate dehydrogenase complex, and this observation has led to a hypothesis that an infection may trigger PBC 12. In addition, another liver disease with unknown etiology, primary sclerosing cholangitis (PSC), is in most patients associated with ulcerative colitis, which may also be triggered by microbial factors 14. Although it has been tempting to speculate about the possible role of helicobacters in chronic inflammatory diseases of the liver, namely PBC, PSC, and chronic autoimmune hepatitis (CAH), the results are conflicting. In a Swedish study, almost all patients with cholestatic end stage PBC and PSC studied with Helicobacter PCR were positive 15, whereas this was shown only for one of 29 American patients suffering from PBC 16. Recently Boomkens et al. 2005 17 found no differences in the presence of Helicobacter DNA in liver biopsies between patients with cholestatic and non‐cholestatic chronic liver diseases. Furthermore, serological studies based on EIAs have failed to show any association between different chronic liver diseases and either H. pylori18,19 or bile‐tolerant Helicobacter species 20. However, when sera were analysed by an immunoblot assay, H. hepaticus was found to be associated with alcoholic liver disease 21 as well as H. pullorum, H. bilis, and H. hepaticus with PSC, PBC and CAH 22,23.

The chronic inflammatory bowel diseases (IBDs), namely ulcerative colitis (UC) and Crohn's disease (CD), are chronic inflammatory diseases of unknown etiology. For both diseases there is a link between several environmental and microbial factors with pathogenesis 14. However, the PCR‐based studies have failed to show any association between helicobacters and IBDs 24,25, and H. pylori DNA in colonic biopsies has been found to occur with the same frequency in IBD as in non‐IBD patients 26–28. Oliveira et al. 2006 29 detected with PCR H. pylori DNA more often in colonic biopsies in a subgroup of Crohn's disease than in non‐IBD patients, but using an EIA method they found H. pylori antibodies to be less common in Crohn's disease than in non‐IBD patients, confirming an earlier finding 30. In four studies non‐pylori helicobacter DNA has been detected by PCR in colonic biopsies, not only in IBD but also in non‐IBD patients 31–34. Thus, despite the attractive hypothesis of Helicobacter infection possibly triggering IBD or autoimmune liver diseases, the scientific evidence remains contradictory.

In this report, in order to assess the role of helicobacters in liver diseases with an emphasis on autoimmune diseases, the presence of Helicobacter DNA in liver specimens of patients with various benign chronic liver diseases was analysed by denaturating gradient gel electrophoresis of PCR products (PCR‐DGGE). The antibody response to immunogenic proteins of Helicobacter species was measured in sera from these patients and in patients with IBD to further study the association between bile‐tolerant helicobacters and these diseases.

Key messages

• Extra‐gastric helicobacters are rare in patients with chronic inflammatory bowel disease or chronic liver disease.

Patients and methods

Patients

Percutaneous liver biopsies taken at Peijas Hospital (Hospital District of Helsinki and Uusimaa) between 1st January 1999 and 30th September 2003 were retrospectively reviewed. Biopsies from patients with malignancy, viral hepatitis, storage diseases of the liver, or known drug‐induced liver damage were excluded. Eighty‐one eligible patients with elevated liver enzymes were identified. From these, 32 patients (median age 56 years, range 27–75; 25 were females) gave serum and stool samples. The serum samples were drawn an average 1.5 years (range 0–8 years) after the liver biopsies had been taken. Twelve patients had PBC, whilst the remainder of the patients had the following clinical diagnoses: CAH in four patients, PSC in one, alcoholic cirrhosis in two, cryptogenic cirrhosis in one, fatty liver in three, focal nodular hyperplasia in one, cholestatic hepatopathy (including two with autoimmune cholangitis) in four, hepatitis with unknown etiology in three, and normal liver in one (in this patient the indication of liver biopsy was slightly elevated liver enzymes). Some characteristics of the patients with liver disease are shown in Table I.

Table I. Characteristics of the 32 patients with liver diseases.

Characteristic	Patients
Females	25
Liver fibrosis grade 0	19/29 ^a
AST mean (reference range 10–35 U/L for females)	47 (range 16–119)
ALT mean (reference range 10–35 U/L for females)	51 (range 8–168)
Alkaline phosphatase mean (reference range 60–275 U/L)	366 (141–1222)
Ursodeoxycholic acid treatment	19

AST = aspartate aminotransferase; ALT = alanine aminotransferase.

^a Three liver specimens were inadequate for the assessment of fibrosis.

The presence of Helicobacter antibodies were studied in serum samples taken from 61 patients suffering from Crohn's disease (median age 37 years, range 17–75 years; median duration of CD 6.3 years, range 0–23 years) and 73 patients suffering from ulcerative colitis (median age 44 years, range 21–77 years; median duration of UC 11.4 years, range 0–44 years). According to the Vienna classification 35 the location of CD was upper gastrointestinal in 6 patients, ileum in 28 patients, ileocolic in 18, and colitis in 9 patients, and the behaviour of CD was penetrating in 12 patients, stricturing in 37 patients, and inflammatory in 12 patients. A total of 37 patients with UC had extensive colitis and 35 had left‐sided colitis.

The Ethics Committee of the Hospital District of Helsinki and Uusimaa approved the study, and all participants gave their informed consent.

Histology

One senior pathologist (JM) experienced in the field of liver pathology reviewed all the liver biopsies and assessed them according to the METAVIR scoring system 36. Standard diagnostic criteria were used to diagnose liver and bowel disease.

Detection of H. pylori and immunoblot assay (IB)

Serum samples were stored at ‐20°C before being analysed. The serum samples from the liver patients and IBD patients were examined by EIA as described earlier 37,38. In the latter H. pylori EIA units <25 were regarded as negative, ⩾35 as positive, and 25–35 as border‐line values. Serum samples from IBD patients with positive H. pylori EIA results were further studied by immunoblot assay (IB).

Stool specimens from the liver patients that were collected at the same time as the serum samples were examined with a polyclonal antibody‐based (Premier Platinum HpSA, Meridian Inc., Cincinnati, OH, USA) and monoclonal antibody‐based (Amplified IDEIA HpStAR test, formerly FemtoLab, Dako, Glostrup, Denmark) stool antigen tests according to the manufacturers' instructions as described earlier 39.

Enteric Helicobacter EIAs and IB

Sera tested for enteric Helicobacter by EIA were initially absorbed with lysed H. pylori cells. The EIAs were performed according to a method recently published 23 with minor modification to the cut‐off values, which were in the present study based on EIA results obtained with sera from 90 healthy Swedish blood donors. Cut‐off values (corresponding to the upper limit for the 95th percentile level of the reference standard and expressed as relative antibody activity) were for H. pullorum ⩾25, for H. bilis ⩾35, and for H. hepaticus ⩾25. Only sera giving positive/border‐line EIA results were further analysed by IB (after absorbing with lysed H. pylori cells) for detection of the three enteric Helicobacter species as described earlier 40.

DNA extraction and amplification conditions

DNA was extracted from paraffin‐embedded liver specimens available for 30 patients as described earlier 9,15 using the QIAamp DNA Kit (QIAGEN, Hilden, Germany).

PCR detection of Helicobacter was done using a nested Helicobacter genus 16S rDNA PCR assay: The primers used in the first step were C97 and C05 41, whereas in the second step they were HG16S 1F with GC‐clamp and HG16S 2R 42.

PCR‐DGGE analysis

Amplified PCR products were analysed in agarose gels containing ethidium bromide and visualized by ultraviolet light. Analysis of Helicobacter‐specific PCR products was done by the DGGE technique as described previously 43. Identification of Helicobacter PCR products was done by comparing the mobility pattern of unknown PCR products with the mobility pattern of PCR products amplified from the following Helicobacter strains: H. muridarum (CCUG 29262), H. bilis (CCUG 38995), H. pullorum (NCTC 12825), H. pylori (CCUG 17874), ‘H. rappini’ (CCUG 23435), H. hepaticus (CCUG 33637), and H. bizzozeronii (AF 53).

16S rDNA sequence analysis

The separated DNA fragments were cut from the DGGE gels and prepared for sequencing as described previously 43. Sequencing of both DNA strands was performed with an ABI 310 DNA sequencer (Applied Biosystems, Foster City, CA, USA), using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.0 (Applied Biosystems). The closest known relatives of the partial 16S rDNA sequences were identified by using BLASTN 2.2.1 (http://www.ncbi.nlm.nih.gov/BLAST/).

Results

Chronic liver disease

Twenty‐three (72%) of the 32 patients with liver disease were negative by H. pylori EIA and by the two different stool antigen tests. Of the nine H. pylori EIA‐positive patients, six were also positive in the two stool antigen tests; from one patient a stool specimen was not available. Positive EIA results were detected for H. bilis in eight patients, for H. hepaticus in nine, and for H. pullorum in seven, and only these were further studied by IB; only one specimen was positive for H. bilis from a patient with autoimmune cholangitis (Table II).

Table II. Positive helicobacter polymerase chain reaction (PCR) results of liver biopsy specimens and serum antibody responses studied by immunoblot (IB) for non‐pylori helicobacters in five patients with different liver diseases. The sera were first screened by enzyme immunoassay (EIA) and only positive samples were further studied by IB.

Patient no.	Clinical diagnosis	EIA positive for	IB positive for	PCR positive for
1	Primary biliary cirrhosis	negative	ND	H. pylori
2	Primary biliary cirrhosis	H. bilis	negative	H. hepaticus
		H. hepaticus	negative
3	Cholestatic hepatitis of unknown etiology	negative	ND	H. pullorum
4	Chronic autoimmune hepatitis	negative	ND	H. pylori H. pullorum H. rappini
5	Autoimmune cholangitis	H. pullorum	H. bilis	negative
		H. bilis
		H. hepaticus

ND = not done.

Of the 30 patients with liver disease with liver specimens suitable for PCR, four had positive Helicobacter PCR results as shown in Table II. The PCR‐DGGE analysis is shown in Figure 1. The patients with positive PCR results were negative in the H. pylori EIA and stool antigen tests, except for the patient positive for H. hepaticus, who was positive for H. pylori Immunoglobulin A (IgA) antibodies.

Figure 1. PCR‐DGGE analysis of 4 Helicobacter positive liver specimens. Lanes: M(mobility)‐Ladder, DNA amplified from reference Helicobacter spp. which consists from top to the bottom of PCR products amplified from H. muridarum (CCUG 29262), H. bilis (CCUG 38995), H. pullorum (NCTC 12825), H. pylori (CCUG 17875), ‘H. rappini’ (CCUG 23435), H. hepaticus (CCUG 33637) and H. bizzozeronii (AF 53). 1: F3A (H. pullorum), F3B (H. pylori), F3C (Helicobacter sp. ‘Flexispira taxon. 8’). 2: F4 (H. pullorum). 3: F11 (H. hepaticus). 4: F17 (H. pylori).

Chronic inflammatory bowel disease

Of the 134 IBD patients, 63 were positive in EIA for H. pylori, and 30 of them were also positive when studied further with IB. The positive EIA and IB results for non‐pylori helicobacters were the following: 24 and 3 for H. bilis, respectively, 48 and 1 for H. hepaticus, respectively, and 32 and 2 for H. pullorum, respectively. Of the CD patients, H. pylori antibodies were found in 13 (21%), H. bilis antibodies in 1 (1.6%), and H. hepaticus antibodies in 1 (1.6%) patient by IB. Of the UC patients, antibodies detected by IB were found in 17 (23%) patients for H. pylori, in 2 (2.7%) for H. bilis, and in 2 (2.7%) for H. pullorum. Certain characteristics of the IBD patients with positive non‐pylori Helicobacter findings are presented in Table III.

Table III. Characteristics of six IBD patients with non‐pylori helicobacter antibodies demonstrated by immunoblot assay. In four of these patients also H. pylori antibodies were detected in the IB.

Patient	Antibodies to Helicobacter sp.	Diagnosis	Age (years)	Duration of IBD (years)	Location and behaviour of IBD
1	H. pylori, H. bilis	CD	57	9	ileum, stricturing
2	H. bilis	UC	29	19	left‐sided colitis
3	H. pylori, H. bilis	UC	34	3	left‐sided colitis
4	H. hepaticus	CD	28	10	ileum, stricturing
5	H. pylori, H. pullorum	UC	50	15	left‐sided colitis
6	H. pylori, H. pullorum	UC	38	6	pancolitis

CD = Crohn's disease; IBD = inflammatory bowel disease; UC = ulcerative colitis.

Discussion

In our study, evidence for non‐pylori Helicobacter species was only rarely found among patients with liver disease and IBDs. We studied the non‐pylori helicobacters in liver disease patients using both IB and PCR methods, and our results were conflicting. Of the 32 liver disease patients only 1 with autoimmune cholangitis had a positive IB result for H. bilis, and of the 30 liver specimens studied by PCR there were only 4 specimens positive for Helicobacters, and none of these patients showed Helicobacter antibodies by IB. Three of these particular patients had autoimmune disease, two had PBC, and one CAH. In the three non‐pylori Helicobacter‐positive cases (of all the four Helicobacter PCR‐positive cases) the PCR was positive for different Helicobacter species, and in one case for three different Helicobacters. Six of the 137 patients with IBD had positive IB results to non‐pylori helicobacters, two with Crohn's disease and four with ulcerative colitis.

Earlier IB‐based studies on non‐pylori helicobacters have shown a high prevalence of these species in patients with autoimmune and alcoholic liver disease 21,22. Although the number of patients in our study was small, all those with alcoholic liver disease, and all except one with autoimmune liver disease, had negative IB results for non‐pylori helicobacters. The limitation in our study is the lack of healthy control patients, but in Sweden, a neighbouring country with very similar infectious disease profiles, the prevalence of non‐pylori helicobacter antibodies among blood donors studied with IB has been shown to be very low. In 4 patients out of 80, non‐pylori helicobacter antibodies were found, and H. hepaticus antibodies were demonstrated in none out of 48 21,22.

An earlier study showing non‐pylori helicobacter PCR products in liver samples from PBC and PSC patients included only patients with end stage liver disease (explanted livers), and an association was also found between the grade of cholestasis and the incidence of Helicobacter15. However, in advanced liver disease, PCR products of several bacteria have often been found 44. In our study, most of the patients had incipient liver disease and the grade of cholestasis in our PBC patients was mild since alkaline phosphatase values were only slightly elevated. Two of the 19 patients with autoimmune liver disease (including patients with PBC, PSC, CAH, and autoimmune cholangitis) had non‐pylori helicobacter PCR products in their liver specimens as compared to 1 of the 13 patients with non‐autoimmune liver disease. In addition, among the PCR positive patients the PCR products belonged to different non‐pylori Helicobacter species. Thus, in accordance with several but not all study reports 15–17, our results do not support the association between Helicobacter species and autoimmune liver diseases.

An earlier serological study investigating the relationship of non‐pylori helicobacters and IBD showed by IB the presence of antibodies to H. pullorum in a subset of patients with UC (10/24) and CD (7/24) 45. However, the Helicobacter PCR studies using colonic biopsies have been negative both in IBD and control patients even for H. pylori PCR products 24,25, or have revealed H. pylori PCR products with almost the same frequency both in IBD and non‐IBD patients 26–28. Oliveira et al. 2006 29 found colonic biopsies to be PCR‐positive for H. pylori more often in a subset of CD patients as compared to the non‐IBD patients. However, in three other studies non‐pylori Helicobacter DNA was found in either a small number of IBD or non‐IBD patients 31–33, the identified species were H. pullorum and H. fennelliae in the German study 32 and H. muridarum and H. cholecystus in the Swedish study 33. In line with these earlier studies we could find by IB antibodies to non‐pylori helicobacters (H. bilis, H. pullorum, or H. hepaticus) in two CD patients and in four UC patients and an association with neither the duration nor the behaviour of these diseases. Thus, both PCR and IB‐based studies have demonstrated only in a few cases a possible association between IBD and non‐pylori helicobacters.

In conclusion, evidence for H. bilis, H. pullorum, or H. hepaticus in patients with chronic but not end stage liver disease and chronic IBD was only rarely detected either by IB or PCR, but we cannot rule out their role in some patients with these diseases.

Acknowledgements

We greatly appreciate the skilful assistance of Pirjo Kosunen and Sari Karesvuori. We are also thankful to Timo Helske MD PhD and Tom Böhling MD PhD for their help during the study.

 This study was supported by grants from the Swedish Science Research Council and Lund University (ALF). This study was partly supported by a grant from City of Helsinki to Lea Veijola. The authors have no financial or other conflicting interest relevant to the study.