The true prevalence of Helicobacter species in the bile of the normal general population is unknown. So far, bile and hepatic specimens have been obtained from patients with pathology, and even controls may not truly reflect the normal population. Data on the correlation between Helicobacter and biliary diseases need to be interpreted with caution.
The hepatobiliary diseases that have been reported to be associated with Helicobacter species in humans range from benign diseases, such as chronic cholecystitis,40 hepatolithiasis,41, 42 primary sclerosing cholangitis12, 43 and primary biliary cirrhosis,44 to malignancies, such as gall-bladder carcinoma40 and primary hepatic carcinomas.45
Helicobacter and biliary diseases
Helicobacter species have been identified in bile and gall-bladder tissue in Chileans with chronic cholecystitis.40 The incidence of carcinoma of the gall-bladder and the biliary tract in Chile is among the highest in the world. In this study, bile and gall-bladder tissue was obtained from Chilean patients with chronic cholecystitis and from controls. Thirteen of 23 bile samples and nine of 23 gall-bladder tissues were positive for Helicobacter. Eight of the Helicobacter-specific PCR fragments were sequenced and subjected to phylogenetic analysis. Five sequences represented strains of H. bilis, two strains of Flexispira rappini and one strain of H. pullorum. In two gall-bladder specimens taken from patients, silver stains detected curved bacteria suggestive of Helicobacter species. The main limitation of this study was that there were only two controls. Both were patients from the USA, one undergoing liver transplantation and one who had died of heart failure. Therefore, the controls were not representative of the patient population, and we cannot exclude the possibility that these organisms are simply part of the normal biota in Chileans. Furthermore, impaired gall-bladder contractility in chronic cholecystitis may alter bile properties to favour the survival of Helicobacter species.
However, a similar study from Mexico, which also has a high incidence of H. pylori infection, failed to detect an association between gallstones and Helicobacter colonization when analysing gall-bladder tissue.46 In this study of patients with cholelithiasis, only one of the 95 gall-bladder specimens was positive for Helicobacter on immunohistochemistry, and only one of 32 by PCR. A study from Germany also produced a negative result.47 Seventy-three bile samples and 11 pancreatic juice samples from patients with acute cholecystitis, chronic cholecystitis or common bile duct occlusion were analysed using 16s rRNA PCR. The test was verified to detect as little as 100 fg of Helicobacter species, and the specificity was determined by southern blotting. None of the bile or pancreatic juice samples showed detectable Helicobacter DNA. Another study found that the prevalence of H. pylori infection, as determined by serology, in 112 patients with gallstones and in 112 controls was no different (82% and 80%, respectively).13
Myung et al. reported that 11% of Korean patients with hepatobiliary diseases had H. pylori infection in bile.41 Seven of 30 patients with hepatolithiasis had evidence of Helicobacter species in bile using PCR, but none of the patients with cholangiocarcinoma, benign strictures, papillomatosis or cystadenocarcinoma, and none of the control group without biliary diseases, had evidence of Helicobacter species in bile. H. pylori DNA was not present in the interior of intrahepatic stones or on the biliary epithelium. Our own data from patients in Hong Kong, who had bile prospectively collected at the time of endoscopic retrograde cholangiography, disclosed that only four of 29 patients with common bile duct stones or cholangitis had Helicobacter species as detected by PCR.48 Neither the two patients with cholangiocarcinoma nor the four controls with normal biliary tracts had Helicobacter genus DNA in their bile. The presence of Helicobacter species DNA in bile did not correlate with the presence of H. pylori in the antrum and the urease B gene was negative in these patients. All patients had received antibiotics prior to endoscopic retrograde cholangiography and culture was negative for Helicobacter species. The results of both of these studies41, 48 do not substantiate a definite role of Helicobacter species in the pathogenesis of biliary diseases, despite the fact that, in both studies, Helicobacter species were only found in patients with cholestasis due to biliary stones. A Japanese study assessed patients with intrahepatic duct stones and found that biliary Campylobacter, rather than the Helicobacter genus, was associated with hepatolithiasis.42Campylobacter is phylogenetically related to the Helicobacter genus, and can inhabit the human upper gastrointestinal tract. Both genera belong to the epsilon branch of the Proteobacter family of Gram-negative bacteria.
Helicobacter species have also been implicated as a cause of primary sclerosing cholangitis. Fox et al. identified Helicobacter species using PCR amplification and southern hybridization in five of eight patients with primary sclerosing cholangitis.12 Subsequent cloning and sequencing showed that these sequences had homology with H. rodentium, Flexispira rappini and H. pullorum. Similar findings of H. pylori DNA by PCR have also been made with regard to primary biliary cirrhosis.44 Confirmation of the bacteria was performed with immunohistochemistry utilizing Helicobacter-specific antibodies and transmission electron microscopy. Nilsson et al., using PCR on liver biopsy specimens, detected Helicobacter species in nine of 12 patients with primary sclerosing cholangitis, 11 of 12 patients with primary biliary cirrhosis and only one of 23 patients with non-cholestatic liver disease or no liver disease.43 Most of these Helicobacter species were shown to be H. pylori by specific primers, southern blot hybridization and sequencing, but not H. bilis, H. pullorum or H. hepaticus. There was no difference in Helicobacter species prevalence between primary sclerosing cholangitis and primary biliary cirrhosis. Among patients with cholestatic diseases, those who had detectable Helicobacter species had a significantly higher alkaline phosphatase level than those without Helicobacter species. This study therefore suggested an association between Helicobacter species and cholestatic diseases, especially in those with higher alkaline phosphatase levels, rather than specifically with either primary sclerosing cholangitis or primary biliary cirrhosis. A relationship between Helicobacter colonization and primary biliary cirrhosis could not be confirmed in another study.49 This study analysed 29 liver specimens from patients with primary biliary cirrhosis for evidence of infection with Helicobacter, mycobacteria, Eubacteria and Archaebacteria using PCR. Archaebacteria and mycobacteria were absent in all cases, and Helicobacter species were identified in only one patient with primary biliary cirrhosis. The difference between these studies may have been the result of the use of different PCR primers. Nilsson et al. detected more cases of H. pylori because two sets of primers were used, one directed against the 16s rRNA region and the other against a 26-kDa protein specific to H. pylori.43
Helicobacter and liver diseases
Several studies have examined the association between Helicobacter species and cirrhosis in patients with chronic liver diseases. Ponzetto et al. examined the seroprevalence of H. pylori in hepatitis C virus (HCV)-infected patients compared with sex- and age-matched controls, and found the prevalences of anti-H. pylori immunoglobulin G antibodies to be 77% and 59%, respectively (P = 0.004).50 Furthermore, analysis of Helicobacter species using PCR targeting the 16s rRNA gene detected Helicobacter species in 23 of 25 liver biopsies from the cirrhotic group. Sequencing showed homology with H. pylori and H. pullorum. The authors concluded that infection with Helicobacter species contributed to the progression of HCV infection. However, the fact that the patients in the control group were not infected with HCV reduces the impact of this study. The progression to cirrhosis may have been due to HCV alone. Longitudinal studies of patients with HCV infection alone compared to those with co-infection with HCV- and bile-resistant Helicobacter species are required to address the issue of whether Helicobacter species are independent risk factors in the development of cirrhosis. Siringo et al. reported that patients with cirrhosis have a higher H. pylori seroprevalence than asymptomatic blood donor controls based on immunoglobulin G serology.51 However, this may be confounded by the older age of the patients in the study group. Nilsson et al. used serology for H. hepaticus and reported that there was no difference in the prevalence of H. hepaticus antibodies between patients with chronic liver disease and controls without liver disease.52
A recent case series found Helicobacter species in eight of eight tumour samples of hepatocellular carcinoma or hepatocholangiocellular carcinoma, compared to only one of eight controls.45 A molecular technique with PCR directed against the 16s rRNA region was used. Sequencing showed that these Helicobacter species were not closely related to H. hepaticus, but rather were as yet unnamed species of hepatic Helicobacter. Histology and culture did not reveal these organisms. This retrospective study used unmatched controls with benign diseases. This raised the possibility that the hepatic carcinomas may have caused chronic intrahepatic cholestasis, with secondary colonization with Helicobacter species. This point was addressed in the study by Nilsson et al., which examined liver tissue surrounding resected primary cholangiocarcinoma or hepatocellular carcinoma and tissue from controls with metastasis from colorectal cancers.53 Twelve of 16 patients with hepatocellular carcinoma, 10 of 14 patients with cholangiocarcinoma but none of the 20 patients with colorectal metastasis had Helicobacter species in the liver samples using PCR. Sequencing of the 16s rDNA fragments showed homology with H. pylori and hepatic Helicobacter species.
Helicobacter and pancreatic diseases
Little is known about the effects of Helicobacter species on the pancreas. Pancreatic juice has antibacterial effects that can be deficient in chronic pancreatitis. In a study of 40 patients with alcoholic chronic pancreatitis, none had detectable H. pylori PCR in the pancreatic juice obtained during endoscopic retrograde cholangiography, despite the presence of H. pylori in the stomach.54 This study suggests that the adverse condition of pancreatic juice inhibits the survival of H. pylori even in the setting of chronic pancreatitis, and therefore it seems unlikely to contribute towards the pathogenesis of pancreatic diseases.
Table 1 illustrates a summary of some of the studies associating Helicobacter and Campylobacter species with hepatobiliary diseases.
Table 1. Studies of Helicobacter and Campylobacter species and association with hepatobiliary diseases
|Reference||Study design||Method of detection||Specimen||Hepatobiliary disease||Helicobacter in subjects||Helicobacter in controls||Organism identified|
|Lin et al.9||Observational||PCR||Bile||Various, including||3/7||No control group||H. pylori|
|(UreA)|| ||malignancies|| || || |
|Fox et al.40||Case–control||PCR||Bile,||Chronic cholecystitis,||13/23||0/2||Helicobacter species|
|(16s rRNA)||gall-bladder||gall-bladder carcinoma|| || ||(H. bilis, Flexispirarappini, H. pullorum)|
|Roe et al.34||Observational||PCR||Bile||CC, pancreatic cancer,||10/32||No control group||Helicobacter species|
|(UreA, 16s rRNA)|| ||hepatolithiasis|| || || |
|Myung et al.41||Case–control||PCR||Bile, biliary||Intrahepatic||7/30||0/13 other biliary||H. pylori|
|(26-Da antigen, UreA)||calculi||ductal calculi|| ||diseases; 0/23 cholecystectomy; 0/8 controls || |
|Nilsson et al.43||Case–control||PCR||Liver||PSC, PBC||9/12* PSC,||0/10 controls,||Helicobacter species|
|(16s rRNA)|| || ||11/12* PBC||1/13 NCLC|| |
|Avenaud et al.45||Case–control||PCR||Liver||Primary hepatic||8/8 primary||1/8||Hepatic Helicobacter|
|(16s rRNA)|| ||carcinoma||hepatic carcinomas|| ||species|
|Nilsson et al.53||Case–control||PCR||Liver||CC, HCC||12/16 CC,||0/20||H. pylori, hepatic|
|(16s rRNA)|| || ||10/14 HCC|| ||Helicobacter species|
|Harada et al.42||Case–control||PCR||Bile, biliary||Intrahepatic||3/14 bile,||0/9 bile,||Campylobacter|
|(16s rRNA)||epithelium||ductal calculi||5/8* biliary epithelium||0/7 biliary epithelium||species (C. rectus, C. showae)|
|Leong et al.48||Case–control||PCR||Bile||CBD stones,||4/25||0/4||Helicobacter species|
|(16s rRNA)|| ||cholangitis|| || || |