| ObjectiveInfection by viruses and parasites can induce chronic inflammation that is a recognized risk factor for cancer in general and especially for carcinoma of the liver. Hepatitis B and hepatitis C viral infections, which induce chronic hepatitis, are major risk factors for hepatocellular carcinoma. The major risk agents for carcinoma of the liver in China are hepatitis viruses. Agents of partial carcinoma and chronic diseases of the liver are not known although new hepatitis viruses are being identified. Bacteria have been linked to cancer only recently. The human pathogen Helicobacter pylori induces a persistent infection that is considered to be a type I carcinogen by World Health Organization in 1994 because of its role in the development of both gastric carcinoma and gastric mucosa-associated lymphoid tissue lymphoma. Also, there are several known Helicobacter species associated with hepatic diseases of animals, such as helicobacter canis in dogs, helicobacter pullorum in poultry, and helicobacter hepaticus in mice. And the latter has been linked to the development of hepatic adenomas and adenocarcinomas in A/JCr mice. Whether Helicobacter species infection can induce hepatobiliary disease or hepatocarcinoma in human? Therefore, identification of humanHelicobacter species in liver is quite important and valuable. The aim of this study is to investigate the relationship between Helicobacter species and primary liver carcinoma.Methods1. First, we chose to study paraffin-embedded liver tissue. Liver samples from 38 patients with primary liver carcinoma diagnosed by histopathology and 15 with liver cirrhosis, 12 with chronic hepatitis, 15 with nonmaligant neoplasm as controls were studied. Helicobacter species in liver specimens from the studied subjects were examined by polymerase chain reaction (PCR) using Helicobacter-specific 16S rRNA gene primers. Amplified products were identified by Southern hybridization and sequencing. A search for other genes specific for Helicobacter pylori was also carried out by PCR. Also, immunohistochemical staining for H. pylori was performed.2. Second, we evaluated the presence of Helicobacter species in fresh liver tissue specimens. Liver samples from 20 patients with primary liver carcinoma diagnosed by histopathology and 16 without as controls were studied. Culture and PCR amplification for Helicobacter-spzcific 16S rRNA gene and H. pylori were used to detect the presence of the bacteria. Amplified products were also identified by Southern hybridization andsequencing.3. Third, we detected Helicobacter species by PCR in peripheral blood of patients with peptic ulcer and H. pylori infection. To test tolerance of H. pylori to bile, bacteria were cultured on blood agar plates containing human bile at different concentration from 0.01%~1.8%.Results1. The detection of Helicobacter species in paraffin-embedded liver tissue: 52.6% (20/38) samples of hepatocellular carcinoma were positive for Helicobacter-specific 16S rRNA gene, while none was positive in control groups (/^O.Ol). Ten of Helicobacter-spQcific PCR amplicons were sequenced and the closet similarity was 99.0%~ 100.0% with 16S rDNA of H pylori. Of these 20 positive samples, 14 were positive with the 26-kDa protein primers of H pylori. The cagA gene was detectable in only 3 samples, and the glmM gene was detectable in 4 samples, while the vacA and rps4 genes were never detectable.2. Immunohistochemical staining for H. pylori in liver tissue specimens: curved or bacillus bacterias were detected in the liver tissue of patients with primary liver carcinoma, the ratio is 42%(16/38). But no bacteria was found in nonmalignant liver tissue.3. Culture: No bacterial growth was detected from cultures of 36 freshliver tissue samples with or without primary liver carcinoma under microaerobic condition.4. The detection of Helicobacter species in fresh liver tissue: 40% (8/20) samples of primary liver carcinoma were posit...
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